All in the Family: Primary Megakaryocytes for Studies of Platelet αIIbβ₃ Signaling

Summary

Integrin αIIbβ3 mediates key platelet adhesive responses during hemostasis and thrombosis. Adhesive ligand binding to αIIbβ3 is regulated by “inside-out” signals, while adhesion-dependent cytoskeletal events are regulated by “outside-in” signals from αIIbβ3. Currently, the molecular basis of bidirectional αIIbβ3 signaling is incompletely understood. The functional assessment of integrin signaling pathways in nucleated cells has been facilitated by techniques such as viral transduction which enable expression of dominant-active and dominant-inhibitory gene products. This approach cannot be used with anucleate platelets. However, recent advances in the ability to expand human and murine megakaryocytes from hematopoietic stem cells provide a tractable and genetically manipulatable system for studies of αIIbβ3 signaling. This overview will discuss some of the advantages and limitations of this approach and provide examples of its utility. Thus, in addition to their intrinsic value for understanding hematopoiesis and platelet formation, primary megakaryocytes represent a model system complementary to platelets for unraveling the remaining mysteries of αIIbβ3 signaling.

Key words

αIIbβ3 - integrin - megakaryocyte - platelet - signaling

• References

• 1 Law DA, Nannizzi-Alaimo L, Cowan KJ, K.S.P Ramakrishnan V, Phillips DR. Signal transduction pathways for mouse platelet membrane adhesion receptors. Thromb Haemost 1999; 82: 345-52.
• 2 Law DA, DeGuzman FR, Heiser P, Ministri-Madrid K, Killeen N, Phillips DR. Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIb-beta3 signalling and platelet function. Nature 1999; 401: 808-11.
• 3 Law DA, Nannizzi-Alaimo L, Ministri K, Hughes P, Forsyth J, Turner M. et al. Genetic and pharmacological analyses of Syk function in α_IIbβ₃ signaling in platelets. Blood 1999; 93: 2645-52.
• 4 Shiraga M, Ritchie A, Aidoudi S, Baron V, Wilcox D, White G. et al. Primary megakaryocytes reveal a role for transcription factor NF-E2 in integrin alpha IIb beta 3 signaling. J Cell Biol 1999; 147: 1419-30.
• 5 Judd BA, Myung PS, Leng L, Obergfell A, Pear WS, Shattil SJ. et al. Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through α_IIbβ₃ and collagen receptors. Proc Natl Acad Sci USA 2000; 97: 12056-61.
• 6 Fox JEB. Platelet activation: New aspects. Haemostasis 1996; 26 (Suppl. 04) 102-31.
• 7 Hartwig JH, Barkalow K, Azim A, Italiano J. The elegant platelet: signals controlling actin assembly. Thromb Haemost 1999; 82: 392-8.
• 8 Shattil SJ. Signaling through platelet integrin α_IIbβ₃: Inside-out, outside-in and sideways. Thromb Haemost 1999; 82: 318-25.
• 9 Dangas G, Badimon JJ, Coller BS, Fallon JT, Sharma SK, Hayes RM. et al. Administration of abciximab during percutaneous coronary intervention reduces both ex vivo platelet thrombus formation and fibrin deposition – Implications for a potential anticoagulant effect of abciximab. Arterioscler Thromb Vasc Biol 1998; 18: 1342-9.
• 10 Calderwood DA, Shattil SJ, Ginsberg MH. Integrins and actin filaments: Reciprocal regulation of cell adhesion and signaling. J Biol Chem 2000; 275: 22607-10.
• 11 Liu S, Calderwood DA, Ginsberg MH. Integrin cytoplasmic domain-binding proteins. J Cell Sci 2000; 113: 3563-71.
• 12 Leisner TM, Wencel-Drake JD, Wang W, Lam SCT. Bidirectional trans-membrane modulation of integrin α_IIbβ₃ conformations. J Biol Chem 1999; 274: 12945-9.
• 13 Vinogradova O, Haas T, Plow EF, Qin J. A structural basis for integrin activation by the cytoplasmic tail of the alpha IIb-subunit. Proc Natl Acad Sci USA 2000; 97: 1450-5.
• 14 Liddington RC, Bankston LA. The structural basis of dynamic cell adhesion: heads, tails, and allostery. Exp Cell Res 2000; 261: 37-43.
• 15 Chen Y-P, Djaffar I, Pidard D, Steiner B, Cieutat A-M, Caen JP. et al. Ser-752→Pro mutation in the cytoplasmic domain of integrin β₃ subunit and defective activation of platelet integrin α_IIbβ₃ (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia. Proc Natl Acad Sci USA 1992; 89: 10169-73.
• 16 Wang R, Shattil SJ, Ambruso DR, Newman PJ. Truncation of the cytoplasmic domain of β₃ in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin α_IIbβ₃ complex. J Clin Invest 1997; 100: 2393-403.
• 17 Liu XY, Timmons S, Lin YZ, Hawiger J. Identification of a functionally important sequence in the cytoplasmic tail of integrin β₃ by using cell-permeable peptide analogs. Proc Natl Acad Sci USA 1996; 93: 11819-24.
• 18 Stephens G, O’Luanaigh N, Reilly D, Harriott P, Walker B, Fitzgerald D. et al. A sequence within the cytoplasmic tail of GPIIb independently activates platelet aggregation and thromboxane synthesis. J Biol Chem 1998; 273: 20317-22.
• 19 Jennings LK, Slack SM, Wall CD, Mondoro TH. Immunological comparisons of integrin alpha IIb beta 3 (GPIIb-IIIa) expressed on platelets and human erythroleukemia cells: evidence for cell specific differences. Blood Cells Mol Dis 1996; 22: 23-35.
• 20 Ody C, Vaigot P, Quéré P, Imhof BA, Corbel C. Glycoprotein IIb-IIIa is expressed on avian multilineage hematopoietic progenitor cells. Blood 1999; 93: 2898-906.
• 21 Tronik-Le Roux D, Roullot V, Poujol C, Kortulewski T, Nurden P, Marguerie G. Thrombasthenic mice generated by replacement of the integrin alpha(IIb) gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment. Blood 2000; 96: 1399-408.
• 22 Handagama P, Scarborough RM, Shuman MA, Bainton DF. Endocytosis of fibrinogen into megakaryocyte and platelet α-granules is mediated by α_IIbβ₃ (glycoprotein IIb-IIIa). Blood 1993; 82: 1358.
• 23 Cheng L, Qasba P, Vanguri P, Thiede MA. Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells. J Cell Physiol 2000; 184: 58-69.
• 24 Blazsek I, Chagraoui J, Peault B. Ontogenic emergence of the hematon, a morphogenetic stromal unit that supports multipotential hematopoietic progenitors in mouse bone marrow. Blood 2000; 96: 3763-71.
• 25 Drachman JG, Sabath DF, Fox NE, Kaushansky K. Thrombopoietin signal transduction in purified murine megakaryocytes. Blood 1997; 89: 483-92.
• 26 Lecine P, Blank V, Shivdasani R. Characterization of the hematopoietic transcription factor NF-E2 in primary murine megakaryocytes. J Biol Chem 1998; 273: 7572-8.
• 27 Lu L, Wang LS, Cooper RJ, Liu HJ, Turner K, Weich N. et al. Suppressive effects of TNF-alpha, TGF-beta1, and chemokines on megakaryocytic colony formation in CD34+ cells derived from umbilical cord blood compared with mobilized peripheral blood and bone marrow. J Hematother Stem Cell Res 2000; 9: 195-204.
• 28 Zauli G, Vitale M, Falcieri E, Gibellini D, Bassini A, Celeghini C. et al. In vitro senescence and apoptotic cell death of human megakaryocytes. Blood 1997; 90: 2234-43.
• 29 Zauli G, Bassini A, Vitale M, Gibellini D, Celeghini C, Caramelli E. et al. Thrombopoietin enhances the α_IIbβ₃-dependent adhesion of megakaryocytic cells to fibrinogen or fibronectin through PI 3 kinase. Blood 1997; 89: 883-95.
• 30 Faraday N, Rade JJ, Johns DC, Khetawat G, Noga SJ, DiPersio JF. et al. Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression. Blood 1999; 94: 4084-92.
• 31 Shiraga M, von Schalscha TLK, Tierney JR, Shattil SJ, Leavitt AD. NF-E2 expression reconstitutes inside-out integrin signaling in NF-E2 p45^-/-megakaryocytes. Blood 2000; 96: 445 (abstr.).
• 32 Wilcox DA, Olsen JC, Ishizawa L, Bray PF, French DL, Steeber DA. et al. Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia. Blood 2000; 95: 3645-51.
• 33 Kovacsovics TJ, Bachelot C, Toker A, Vlahos CJ, Duckworth B, Cantley LC. et al. Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. J Biol Chem 1995; 270: 11358-66.
• 34 Rittenhouse SE. Phosphoinositide 3-kinase activation and platelet function. Blood 1996; 88: 4401-14.
• 35 Poujol C, Nurden AT, Nurden P. Ultrastructural analysis of the distribution of the vitronectin receptor (alpha v beta 3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the alpha-granule membrane. Br J Haematol 1997; 96: 823-35.
• 36 Coulombel L, Auffray I, Gaugler MH, Rosemblatt M. Expression and function of integrins on hematopoietic progenitor cells. Acta Haematol 1997; 97: 13-21.
• 37 Arroyo AG, Yang JT, Rayburn H, Hynes RO. Alpha4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo. Immunity 1999; 11: 555-66.
• 38 Molla A, Mossuz P, Berthier R. Extracellular matrix receptors and the differentiation of human megakaryocytes in vitro. Leuk Lymphoma 1999; 33: 15-23.
• 39 Potocnik AJ, Brakebusch C, Fassler R. Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow. Immunity 2000; 12: 653-63.
• 40 Sakai T, Zhang QH, Fässler R, Mosher DF. Modulation of β1A integrin functions by tyrosine residues in the β₁ cytoplasmic domain. J Cell Biol 1998; 141: 527-38.
• 41 Rose DM, Cardarelli PM, Cobb RR, Ginsberg MH. Soluble VCAM-1 binding to 4 integrins is cell-type specific and activation dependent and is disrupted during apoptosis in T cells. Blood 2000; 95: 602-9.
• 42 Byzova TV, Goldman CK, Pampori N, Thomas KA, Bett A, Shattil SJ. et al. A mechanism for modulation of cellular responses to VEGF: Activation of the integrins. Molecular Cell 2000; 6: 851-60.
• 43 Kahn ML, Zheng YW, Huang W, Bigornia V, Zeng DW, Moff S. et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-4.
• 44 Mountford JC, Melford SK, Bunce CM, Gibbons J, Watson SP. Collagen or collagen-related peptide cause [Ca 2+ ]i elevation and increased tyrosine phosphorylation in human megakaryocytes. Thromb Haemost 1999; 82: 1153-9.
• 45 Rojnuckarin P, Drachman JG, Kaushansky K. Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: Role in endomitosis. Blood 1999; 94: 1273-82.
• 46 Wang Q, Miyakawa Y, Fox N, Kaushansky K. Interferon- directly represses megakaryopoiesis by inhibiting thrombopoietin-induced signaling through induction of SOCS-1. Blood 2000; 96: 2093-9.
• 47 Leven RM, Nachmias VT. Cultured megakaryocytes: changes in the cyto-skeleton after adenosine diphosphate-induced spreading. J Cell Biol 1982; 92: 313-23.
• 48 Leng L, Kashiwagi H, Ren X-D, Shattil SJ. RhoA and the function of platelet integrin α_IIbβ₃ . Blood 1998; 91: 4206-15.
• 49 Sastry SK, Burridge K. Focal adhesions: A nexus for intracellular signaling and cytoskeletal dynamics. Exp Cell Res 2000; 261: 25-36.
• 50 Lane WJ, Dias S, Hattori K, Heissig B, Choy M, Rabbany SY. et al. Stromal-derived factor 1-induced megakaryocyte migration and platelet production is dependent on matrix metalloproteinases. Blood 2000; 96: 4152-9.
• 51 Italiano Jr JE, Lecine P, Shivdasani RA, Hartwig JH. Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes. J Cell Biol 1999; 147: 1299-312.
• 52 Rojnuckarin P, Kaushansky K. Actin reorganization and proplatelet formation in murine megakaryocytes: the role of protein kinase Ca. Blood 2001; 97: 154-61.
• 53 Takahashi R, Sekine N, Nakatake T. Influence of monoclonal antiplatelet glycoprotein antibodies on in vitro human megakaryocyte colony formation and proplatelet formation. Blood 1999; 93: 1951-8.
• 54 Hodivala-Dilke KM, McHugh KP, Tsakiris DA, Rayburn H, Crowley D, Ullman-Cullere M. et al. Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J Clin Invest 1999; 103: 229-38.
• 55 Thompson A, Zhang Y, Kamen D, Jackson CW, Cardiff RD, Ravid K. Deregulated expression of c-myc in megakaryocytes of transgenic mice increases megakaryopoiesis and decreases polyploidization. J Biol Chem 1996; 271: 22976-82.
• 56 Tronik-Le Roux D, Roullot V, Schweitzer A, Berthier R, Marguerie G. Suppression of erythro-megakaryocytopoiesis and the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase gene targeted by the platelet glycoprotein alpha IIb promoter. J Exp Med 1995; 181: 2141-51.
• 57 Murphy GJ, Leavitt AD. A model for studying megakaryocyte development and biology. Proc Natl Acad Sci USA 1999; 96: 3065-70.
• 58 Castellino SM, Kurtzberg J, Smith C. Retroviral vector-mediated gene transfer into umbilical cord blood- derived megakaryocyte and platelet progenitors. Biol Blood Marrow Transplant 1999; 5: 215-21.
• 59 Lecine P, Italiano Jr JE, Kim SW, Villeval JL, Shivdasani RA. Hematopoietic-specific beta 1 tubulin participates in a pathway of platelet biogenesis dependent on the transcription factor NF-E2. Blood 2000; 96: 1366-73.
• 60 Wilcox DA, Olsen JC, Ishizawa L, Griffith M, White GC. 2nd. Integrin alphaIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells. Proc Natl Acad Sci USA 1999; 96: 9654-9.
• 61 Melford SK, Turner M, Briddon SJ, Tybulewicz VL, Watson SP. Syk and Fyn are required by mouse megakaryocytes for the rise in intracellular calcium induced by a collagen-related peptide. J Biol Chem 1997; 272: 27539-42.
• 62 Pasquet JM, Bobe R, Gross B, Gratacap MP, Tomlinson MG, Payrastre B. et al. A collagen-related peptide regulates phospholipase Cgamma2 via phosphatidylinositol 3-kinase in human platelets. Biochem J 1999; 342: 171-7.
• 63 Briddon SJ, Melford SK, Turner M, Tybulewicz V, Watson SP. Collagen mediates changes in intracellular calcium in primary mouse megakaryocytes through syk-dependent and -independent pathways. Blood 1999; 93: 3847-55.
• 64 Gaur M, Murphy GJ, DeSauvage FJ, Leavitt AD. Thrombopoietin receptor (cMPL) structure-function studies using primary megakaryocyte lineage cells from MPL^-/- mice. Blood 2000; 96: 567 (abstr.).
• 65 Wilcox DA, Hodivala-Dilke KM, Steeber DA, Shattil SJ, Hynes RO, White GC. Expression of a functional murine αIIb-human β₃ heterodimer complex on the surface of megakaryocytes derived from β₃-knockout mice. Thromb Haemost 1999; 82: 1168 (abstr.).
• 66 Chen Y-P, O’Toole TE, Shipley T, Forsyth J, LaFlamme SE, Yamada KM. et al. “Inside-out” signal transduction inhibited by isolated integrin cytoplasmic domains. J Biol Chem 1994; 269: 18307-10.
• 67 Kashiwagi H, Schwartz MA, Eigenthaler MA, Davis KA, Ginsberg MH, Shattil SJ. Affinity modulation of platelet integrin α_IIbβ₃ by β₃-endonexin, a selective binding partner of the β₃ integrin cytoplasmic tail. J Cell Biol 1997; 137: 1433-43.
• 68 Schwarze SR, Hruska KA, Dowdy SF. Protein transduction: unrestricted delivery into all cells?. Trends Cell Biol 2000; 10: 290-5.
• 69 Tsakiris DA, Scudder L, Hodivala-Dilke K, Hynes RO, Coller BS. Hemostasis in the mouse (Mus musculus): A review. Thromb Haemost 1999; 81: 177-88.
• 70 Hoying JB, Yin M, Diebold R, Ormsby I, Becker A, Doetschman T. Transforming growth factor beta1 enhances platelet aggregation through a nontranscriptional effect on the fibrinogen receptor. J Biol Chem 1999; 274: 31008-13.
• 71 Hauser W, Knobeloch KP, Eigenthaler M, Gambaryan S, Krenn V, Geiger J. et al. Megakaryocyte hyperplasia and enhanced agonist-induced platelet activation in vasodilator-stimulated phosphoprotein knockout mice. Proc Natl Acad Sci USA 1999; 96: 8120-5.
• 72 Vyas P, Ault K, Jackson CW, Orkin SH, Shivdasani RA. Consequences of GATA-1 deficiency in megakaryocytes and platelets. Blood 1999; 93: 2867-75.
• 73 Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, Jackson CW, Hunt P, Saris CJ. et al. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell 1995; 81: 695-704.
• 74 Levin J, Peng JP, Baker GR, Villeval JL, Lecine P, Burstein SA. et al. Pathophysiology of thrombocytopenia and anemia in mice lacking transcription factor NF-E2. Blood 1999; 94: 3037-47.
• 75 Deveaux S, Cohen-Kaminsky S, Shivdasani RA, Andrews NC, Filipe A, Kuzniak I. et al. p45 NF-E2 regulates expression of thromboxane synthase in megakaryocytes. Embo J 1997; 16: 5654-61.
• 76 Vitrat N, Letestu R, Masse A, Lazar V, Vainchenker W, Debili N. Thromboxane synthase has the same pattern of expression as platelet specific glycoproteins during human megakaryocyte differentiation. Thromb Haemost 2000; 83: 759-68.
• 77 Coughlin SR. Thrombin signalling and protease-activated receptors. Nature 2000; 407: 258-64.
• 78 Smyth SS, Tsakiris DA, Scudder LE, Coller BS. Structure and function of murine α_IIbβ₃ (GPIIb/IIIa): Studies using monoclonal antibodies and β_3-null mice. Thromb Haemost 2000; 84: 1103-8.