Download PDF
Thromb Haemost 2001; 86(04): 1106-1113
DOI: 10.1055/s-0037-1616540
Special Article
Schattauer GmbH

Biogenesis of G-protein Mediated Calcium Signaling in Human Megakaryocytes

Els den Dekker
1   Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht and Institute for Biomembranes, Utrecht University, The Netherlands
,
Gertie Gorter
1   Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht and Institute for Biomembranes, Utrecht University, The Netherlands
,
Hans van der Vuurst
1   Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht and Institute for Biomembranes, Utrecht University, The Netherlands
,
Johan W. M. Heemskerk
2   Departments of Biochemistry and Human Biology, Maastricht University, The Netherlands
,
Jan-Willem N. Akkerman
1   Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht and Institute for Biomembranes, Utrecht University, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 16 January 2000

Accepted after revision 16 May 2001

Publication Date:
09 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

To understand how platelet signal transduction pathways develop during megakaryocytopoiesis, we isolated human stem cells from umbilical cord blood and cultured the cells in the presence of thrombopoietin (TPO). Based on the early expression of CD61 and late expression of CD42b, immature (CD61+/CD42blow) and mature (CD61+/ CD42bhigh) megakaryocytes were immunomagnetically purified and, together with stem cells (CD34+), characterized for G -protein expression and agonist-induced [Ca2+]i increases. Megakaryocytopoiesis was accompanied by down-regulation of the 43 kDa and 46 kDa variants of G16α, constant expression of Gsα, and up-regulation of Gqα and Giα1/2. The increase in Gq and Gi 1/2 expression was accompanied by an increase in Ca2+ signaling triggered by thrombin and other agonists known to signal to Ca2+ via these G-proteins in platelets. The prostacyclin analog iloprost and TPO also induced [Ca2+]i increases, and the iloprost-induced Ca2+ response disappeared during maturation. These data reveal sharp changes in Ca2+ regulation during megakaryocytopoiesis.

Keywords

Megakaryocytopoiesis - signaling - trimeric G-proteins - calcium
 

  • References

  • 1 Siess W. Molecular mechanisms of platelet activation. Physiol Rev 1989; 69: 58-178.
    CrossrefPubMedGoogle Scholar
  • 2 Offermanns S. The role of heterotrimeric G proteins in platelet activation. Biol Chem 2000; 381: 389-96.
    PubMedGoogle Scholar
  • 3 Doni MG, Cavallini L, Alexandre A. Ca2+ influx in platelets: activation by thrombin and by the depletion of the stores. Effect of cyclic nucleotides. Biochem J 1994; 303: 599-605.
    CrossrefPubMedGoogle Scholar
  • 4 Knight DE, Scrutton MC. Cyclic nucleotides control a system which regulates Ca2+ sensitivity of platelet secretion. Nature 1984; 309: 66-8.
    CrossrefPubMedGoogle Scholar
  • 5 Watson SP, McConnell RT, Lapetina EG. The rapid formation of inositol phosphates in human platelets by thrombin is inhibited by prostacyclin. J Biol Chem 1984; 259: 13199-203.
    PubMedGoogle Scholar
  • 6 Rao AK, Gabbeta J. Congenital disorders of platelet signal transduction. Arterioscler Thromb Vasc Biol 2000; 20: 285-9.
    CrossrefPubMedGoogle Scholar
  • 7 Rao AK, Kowalska MA, Disa J. Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects. Blood 1989; 74: 664-72.
    PubMedGoogle Scholar
  • 8 Rao AK, Disa J, Yang X. Concomitant defect in internal release and influx of calcium in patients with congenital platelet dysfunction and impaired agonist-induced calcium mobilization. Thromboxane production is not required for internal release of calcium. J Lab Clin Med 1993; 121: 52-63.
    PubMedGoogle Scholar
  • 9 van der Vuurst H, van Willigen G, van Spronsen A, Hendriks M, Donath J, Akkerman JWN. Signal transduction through trimeric G proteins in megakaryoblastic cell lines. Arterioscler Thromb Vasc Biol 1997; 17: 1830-6.
    CrossrefPubMedGoogle Scholar
  • 10 de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 1994; 369: 533-8.
    CrossrefPubMedGoogle Scholar
  • 11 Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort PJ, Grant FJ, Heipel MD, Burkhead SK, Kramer JM. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 1994; 369: 565-8.
    CrossrefPubMedGoogle Scholar
  • 12 Debili N, Wendling F, Katz A, Guichard J, Breton-Gorius J, Hunt P, Vainchenker W. The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors. Blood 1995; 86: 2516-25.
    PubMedGoogle Scholar
  • 13 Cohen-Solal K, Debili N, Vainchenker W, Wendling F. Thrombopoietin (Mpl-ligand) and the regulation of platelet production. Thromb Haemost 1997; 78: 37-41.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Debili N, Issaad C, Masse JM, Guichard J, Katz A, Breton-Gorius J, Vainchenker W. Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation. Blood 1992; 80: 3022-35.
    PubMedGoogle Scholar
  • 15 Zucker-Franklin D, Kaushansky K. Effect of thrombopoietin on the development of megakaryocytes and platelets: an ultrastructural analysis. Blood 1996; 88: 1632-8.
    PubMedGoogle Scholar
  • 16 Cramer EM, Norol F, Guichard J, Breton-Gorius J, Vainchenker W, Masse JM, Debili N. Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand. Blood 1997; 89: 2336-46.
    PubMedGoogle Scholar
  • 17 Berridge MJ. Calcium signalling and cell proliferation. Bioessays 1995; 17: 491-500.
    CrossrefPubMedGoogle Scholar
  • 18 Magocsi M, Apati A, Gati R, Kolonics A. Signalling mechanisms and the role of calcineurin in erythropoiesis. Immunol Lett 1999; 68: 187-95.
    CrossrefPubMedGoogle Scholar
  • 19 Haslauer M, Baltensperger K, Porzig H. Erythropoietin- and stem cell factor-induced DNA synthesis in normal human erythroid progenitor cells requires activation of protein kinase Cα and is strongly inhibited by thrombin. Blood 1999; 94: 114-26.
    PubMedGoogle Scholar
  • 20 Heemskerk JWM, Vuist WMJ, Feijge MAH, Reutelingsperger CPM, Lind-hout T. Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses. Blood 1997; 90: 2615-25.
    PubMedGoogle Scholar
  • 21 Keularts IMLW, van Gorp RMA, Feijge MAH, Vuist WMJ, Heemskerk JWM. α2A-adrenergic receptor stimulation potentiates calcium release in platelets by modulating cAMP levels. J Biol Chem 2000; 275: 1763-72.
    CrossrefPubMedGoogle Scholar
  • 22 Tenailleau S, Corre I, Hermouet S. Specific expression of heterotrimeric G proteins G12 and G16 during human myeloid differentiation. Exp Hematol 1997; 25: 927-34.
    PubMedGoogle Scholar
  • 23 Banno Y, Asano T, Nozawa Y. Stimulation by G protein βγ subunits of phospholipase Cβ isoforms in human platelets. Thromb Haemost 1998; 79: 1008-13.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 24 Giesberts AN, van Ginneken M, Gorter G, Lapetina EG, Akkerman JWN, van Willigen G. Subcellular localization of α-subunits of trimeric G-proteins in human platelets. Biochem Biophys Res Commun 1997; 234: 439-44.
    CrossrefPubMedGoogle Scholar
  • 25 Rhee SG, Bae YS. Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem 1997; 272: 15045-8.
    CrossrefPubMedGoogle Scholar
  • 26 Crouch MF, Lapetina EG. A role for Gi in control of thrombin receptor-phospholipase C coupling in human platelets. J Biol Chem 1988; 263: 3363-71.
    PubMedGoogle Scholar
  • 27 Siess W, Weber PC, Lapetina EG. Activation of phospholipase C is dissociated from arachidonate metabolism during platelet shape change induced by thrombin or platelet-activating factor. Epinephrine does not induce phospholipase C activation or platelet shape change. J Biol Chem 1984; 259: 8286-92.
    PubMedGoogle Scholar
  • 28 Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 1998; 95: 8070-4.
    CrossrefPubMedGoogle Scholar
  • 29 Pulcinelli FM, Ciampa MT, Favilla M, Pignatelli P, Riondino S, Gazzaniga PP. Concomitant activation of Gi protein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation. FEBS Lett 1999; 460: 37-40.
    CrossrefPubMedGoogle Scholar
  • 30 Paul BZ, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A2-induced platelet aggregation. Essential role for P2tac and α2a receptors. J Biol Chem 1999; 274: 29108-14.
    CrossrefPubMedGoogle Scholar
  • 31 Offermanns S, Toombs CF, Hu YH, Simon MI. Defective platelet activation in Gαq-deficient mice. Nature 1997; 389: 183-6.
    CrossrefPubMedGoogle Scholar
  • 32 van der Vuurst H, Hendriks M, Lapetina EG, van Willigen G, Akkerman JWN. Maturation of megakaryoblastic cells is accompanied by upregulation of Gsα-L subtype and increased cAMP accumulation. Thromb Haemost 1998; 79: 1014-20.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 Andreopoulos S, Li PP, Siu KP, Warsh JJ. Characterization of αs-immuno-reactive ADP-ribosylated proteins in postmortem human brain. J Neurosci Res 1999; 56: 632-43.
    CrossrefPubMedGoogle Scholar
  • 34 Molina y Vedia L, Nolan RD, Lapetina EG. The effect of iloprost on the ADP-ribosylation of Gsα (the alpha-subunit of Gs). Biochem J 1989; 261: 841-5.
    CrossrefPubMedGoogle Scholar
  • 35 Amatruda TT, Steele DA, Slepak VZ, Simon MI. Gα16, a G protein α subunit specifically expressed in hematopoietic cells. Proc Natl Acad Sci USA 1991; 88: 5587-91.
    CrossrefPubMedGoogle Scholar
  • 36 Baltensperger K, Porzig H. The P2U purinoceptor obligatorily engages the heterotrimeric G protein G16 to mobilize intracellular Ca2+ in human erythroleukemia cells. J Biol Chem 1997; 272: 10151-9.
    CrossrefPubMedGoogle Scholar
  • 37 Wu D, LaRosa GJ, Simon MI. G protein-coupled signal transduction pathways for interleukin-8. Science 1993; 261: 101-3.
    CrossrefPubMedGoogle Scholar
  • 38 Higuchi T, Koike K, Sawai N, Mwamtemi HH, Takeuchi K, Shiohara M, Kikuchi T, Yasui K, Ito S, Yamagami O, Sasaki Y, Okumura N, Kato T, Miyazaki H, Ikeda M, Yamada M, Komiyama A. Megakaryocytes derived from CD34-positive cord blood cells produce interleukin-8. Br J Haematol 1997; 99: 509-16.
    CrossrefPubMedGoogle Scholar
  • 39 Gewirtz AM, Zhang J, Ratajczak J, Ratajczak M, Park KS, Li C, Yan Z, Poncz M. Chemokine regulation of human megakaryocytopoiesis. Blood 1995; 86: 2559-67.
    PubMedGoogle Scholar
  • 40 Tertyshnikova S, Fein A. Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell. Proc Natl Acad Sci USA 1998; 95: 1613-7.
    CrossrefPubMedGoogle Scholar
  • 41 Heemskerk JWM, Vis P, Feijge MAH, Hoyland J, Mason WT, Sage SO. Roles of phospholipase C and Ca2+-ATPase in calcium responses of single, fibrinogen-bound platelets. J Biol Chem 1993; 268: 356-63.
    PubMedGoogle Scholar
  • 42 Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelet paradigm. Blood 1998; 91: 2645-57.
    PubMedGoogle Scholar
  • 43 Shiraga M, Ritchie A, Aidoudi S, Baron V, Wilcox D, White G, Ybarrondo B, Murphy G, Leavitt A, Shattil S. Primary megakaryocytes reveal a role for transcription factor NF-E2 in integrin αIIbβ3 signaling. J Cell Biol 1999; 147: 1419-30.
    CrossrefPubMedGoogle Scholar
  • 44 Schwaner I, Seifert R, Schultz G. Receptor-mediated increases in cytosolic Ca2+ in the human erythroleukaemia cell line involve pertussis toxin-sensitive and -insensitive pathways. Biochem J 1992; 281: 301-7.
    CrossrefPubMedGoogle Scholar
  • 45 Sasaki Y, Takahashi T, Tanaka I, Nakamura K, Okuno Y, Nakagawa O, Narumiya S, Nakao K. Expression of prostacyclin receptor in human megakaryocytes. Blood 1997; 90: 1039-46.
    PubMedGoogle Scholar
  • 46 Kunitama M, Shimizu R, Yamada M, Kato T, Miyazaki H, Okada K, Miura Y, Komatsu N. Protein kinase C and c-myc gene activation pathways in thrombopoietin signal transduction. Biochem Biophys Res Commun 1997; 231: 290-4.
    CrossrefPubMedGoogle Scholar