Download PDF
Thromb Haemost 2000; 84(02): 312-318
DOI: 10.1055/s-0037-1614013
Review Article
Schattauer GmbH

Ultrastructural Analysis of Megakaryocytes in GPV Knockout Mice

Christel Poujol
2   From UMR 5533 CNRS, Hôpital Cardiologique, Pessac, France
,
Vanitha Ramakrishnan
1   Cor Therapeutics, South San Francisco, California, USA
,
Francis DeGuzman
1   Cor Therapeutics, South San Francisco, California, USA
,
Alan T. Nurden
2   From UMR 5533 CNRS, Hôpital Cardiologique, Pessac, France
,
David R. Phillips
1   Cor Therapeutics, South San Francisco, California, USA
,
Paquita Nurden
2   From UMR 5533 CNRS, Hôpital Cardiologique, Pessac, France
› Author AffiliationsC.P. was the recipient of a post-doctoral fellowship from the Institut Electricité Santé.
Further Information

Publication History

Received 06 January 2000

Accepted after revision 21 March 2000

Publication Date:
14 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

Lesions in the genes for GPIbα, GPIbβ or GPIX result in a bleeding diathesis, the Bernard-Soulier syndrome (BSS), which associates a platelet adhesion defect with thrombocytopenia, giant platelets and abnormal megakaryocytes (MK). The role of GPV, also absent in BSS, was recently addressed by gene targeting in mice. While a negative modulator function for GPV on thrombin-induced platelet responses was found in one model, the absence of GP V had no effect on GPIb-IX expression or platelet adhesion. Our study extends previous results and reports that electron microscopy of bone marrow from the GPV knockout mice revealed a normal MK ultrastructure and development of the demarcation membrane system (DMS). There was a usual presence of MK fragments in the bone marrow vascular sinus. Immunogold labelling of MK from the knockout mice showed a normal distribution of GPIb-IX in the DMS and on the cell surface. The distribution of fibrinogen, vWF and P-selectin was unchanged with, interestingly, P-selectin also localised within the DMS in both situations. Thus GPV is not crucial to MK development and platelet production, consistent with the fact that no mutation in the GPV gene has as yet been described in BSS.

Key words

GPV knockout mice - GPIb-IX - megakaryocytes - electron microscopy
 

  • References

  • 1 Lopez JA, Afshar-Kharghan V, Berndt MC. Bernard-Soulier syndrome. Blood 1998; 91: 4397-418.
    PubMedGoogle Scholar
  • 2 Ware J. Molecular analyses of the platelet glycoprotein Ib-IX-V receptor. Thromb Haemost 1998; 79: 466-78.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 1998; 94: 657-66.
    CrossrefPubMedGoogle Scholar
  • 4 Andrews RK, Lopez JA, Berndt MC. Molecular mechanisms of platelet adhesion and activation. Int J Biochem Cell Biol 1997; 29: 91-105.
    CrossrefPubMedGoogle Scholar
  • 5 George JN, Nurden AT, Phillips DR. Molecular defects in interactions of platelets with the vessel wall. N Engl J Med 1984; 311: 1084-98.
    CrossrefPubMedGoogle Scholar
  • 6 Clemetson KJ, Clemetson JM. Molecular abnormalities in Glanzmann’s thrombasthenia, Bernard-Soulier syndrome, and platelet-type von Willebrand disease. Current Opinion Hematol 1994; 01: 388-93.
    PubMedGoogle Scholar
  • 7 Nurden AT, George JN. Inherited disorders of the platelet membrane: Glanzmann thrombasthenia, Bernard-Soulier syndrome and other disorders. In: Colman RW, Hirsh J, Marder VJ, Salzman EW. (eds). Hemostasis and Thrombosis. Basic Principles and Clinical Practice. Vol IV. Philadelphia: JB Lippincott Co; In Press.
    Google Scholar
  • 8 Nurden P, Nurden AT. Giant platelets, megakaryocytes and the expression of glycoprotein Ib-IX complexes. C R Acad Sci 1996; 319: 717-26.
    PubMedGoogle Scholar
  • 9 Lopez JA, Leung B, Reynold CC, Li CQ, Fox JE. Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits. J Biol Chem 1992; 267: 12851-9.
    PubMedGoogle Scholar
  • 10 Dong J, Gao S, Lopez JA. Synthesis, assembly and intracellular transport of the platelet glycoprotein Ib-IX-V complex. J Biol Chem 1998; 273: 31449-54.
    CrossrefPubMedGoogle Scholar
  • 11 Meyer SC, Fox JEB. Interaction of platelet glycoprotein V with glycoprotein Ib-IX regulates expression of the glycoproteins and binding of von Willebrand factor to glycoprotein Ib-IX in transfected cells. J Biol Chem 1995; 270: 14693-9.
    CrossrefPubMedGoogle Scholar
  • 12 Calverley DC, Yagi M, Stray SM, Roth GJ. Human platelet glycoprotein V: its role in enhancing expression of the glycoprotein Ib receptor. Blood 1995; 86: 1361-7.
    PubMedGoogle Scholar
  • 13 Phillips DR, Poh PAgin. Platelet plasma membrane glycoproteins. Identification of a proteolytic substrate for thrombin. Biochem Biophys Res Commun 1977; 75: 940-7.
    CrossrefPubMedGoogle Scholar
  • 14 Bienz D, Schnippering W, Clemetson KJ. Glycoprotein V is not the thrombin activation receptor on human blood platelets. Blood 1986; 68: 720-5.
    PubMedGoogle Scholar
  • 15 Ramakrishnan V, Reeves PS, DeGuzman F, Deshpande U, Ministri-Madrid K, DuBridge RB, Phillips DR. Increased thrombin responsiveness in platelets from mice lacking glycoprotein V. Proc Natl Acad Sci USA 1999; 96: 13336-41.
    CrossrefPubMedGoogle Scholar
  • 16 Kahn ML, Diacovo TG, Bainton DF, Lanza F, Trejo JA, Coughlin SR. Glycoprotein V-deficient platelets have undiminished thrombin responsiveness and do not exhibit a Bernard-Soulier phenotype. Blood 1999; 94: 4112-21.
    PubMedGoogle Scholar
  • 17 Romo GM, Dong JF, Schade AJ, Gardiner EE, Kansas GS, Li CQ, McIntire LV, Berndt MC, Lopez JA. The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin. J Exp Med 1999; 190: 803-14.
    CrossrefPubMedGoogle Scholar
  • 18 Poujol C, Tronik-Le Roux D, Tropel P, Roullot V, Nurden AT, Marguerie G, Nurden P. Ultrastructural analysis of bone marrow hematopoiesis in mice transgenic for the thymidine kinase gene driven by the αIIb promoter. Blood 1998; 92: 2012-23.
    PubMedGoogle Scholar
  • 19 Hürlimann-Forster M, Steiner B, von Felten A. Quantitation of platelet-specific autoantibodies in platelet eluates of ITP patients measured by a novel ELISA using the purified glycoprotein complexes GPIIb/IIIa and GPIb/IX as antigens. Brit J Haematol 1997; 98: 328-35.
    CrossrefPubMedGoogle Scholar
  • 20 Hourdillé P, Pico MJandrot-Perrus, Lacaze D, Lozano M, Nurden AT. Studies on the megakaryocytes of a patient with the Bernard-Soulier syndrome. Brit J Haematol 1990; 76: 521-30.
    CrossrefPubMedGoogle Scholar
  • 21 Berger G, Massé J-M, Cramer EM. Alpha-granule membrane mirrors the platelet plasma membrane and contains the glycoproteins Ib, IX and V. Blood 1996; 87: 1385-95.
    PubMedGoogle Scholar
  • 22 Modderman PW, Admiraal LG, Sonnenberg A, von dem Borne AE. Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane. J Biol Chem 1992; 267: 364-9.
    PubMedGoogle Scholar
  • 23 Sporn LA, Chavin SI, Marder VJ, Wagner DD. Biosynthesis of von Willebrand protein by human megakaryocytes. J Clin Invest 1985; 76: 1102-6.
    CrossrefPubMedGoogle Scholar
  • 24 Harrison P, Wilbourn B, Debili N, Vainchenker W, Breton-Gorius J, Lawrie AS, Masse J-M, Savidge GF, Cramer EM. Uptake of plasma fibrinogen into the alpha granules of human megakaryocytes and platelets. J Clin Invest 1989; 84: 1320-4.
    CrossrefPubMedGoogle Scholar
  • 25 Cramer EM, Vainchenker W, Vinci G, Guichard J, Breton-Gorius J. Gray platelet syndrome: immunoelectron microscopic localization of fibrinogen and von Willebrand factor in platelets and megakaryocytes. Blood 1985; 66: 1309-16.
    PubMedGoogle Scholar
  • 26 Nurden P, Poujol C, Durrieu-Jais C, Winckler J, Combrié R, Macchi L, Bihour C, Wagner C, Jordan R, Nurden AT. Labelling of the internal pool of GP IIb-IIIa in platelets by c7E3 Fab fragments (abciximab): flow and endocytic mechanisms contribute to the transport. Blood 1999; 93: 1622-33.
    PubMedGoogle Scholar
  • 27 Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet α-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol 1985; 101: 880-6.
    CrossrefPubMedGoogle Scholar
  • 28 Tomer A, Scharf RE, McMillan R, Ruggeri ZM, Harker LA. Bernard-Soulier syndrome: quantitative characterization of megakaryocytes and platelets by flow cytometric and platelet kinetic measurements. Eur J Haematol 1994; 52: 193-200.
    PubMedGoogle Scholar
  • 29 Ware J, Russell SR, Marchese P, Murata M, Mazzucato M, De Marco L, Ruggeri ZM. Point mutation in a leucine-rich repeat of platelet glycoprotein Ibα resulting in the Bernard-Soulier syndrome. J Clin Invest 1993; 92: 1213-22.
    CrossrefPubMedGoogle Scholar
  • 30 Miller JL, Lyle VA, Cunningham D. Mutation of leucine-57 to phenylalanine in a platelet glycoprotein Ibα leucine tandem repeat occurring in patients with an autosomal dominant variant of Bernard-Soulier disease. Blood 1992; 79: 439-46.
    PubMedGoogle Scholar
  • 31 Lanza F, Morales M, De la Salle C, Cazenave J-P, Clemetson KJ, Shimomura T, Phillips DR. Cloning and characterization of the gene encoding the human platelet glycoprotein V. A member of the leucine-rich glycoprotein family cleaved during thrombin-induced platelet activation. J Biol Chem 1993; 268: 20801-7.
    PubMedGoogle Scholar
  • 32 Lepage A, Uzan G, Touche N, Morales M, Cazenave JP, Lanza F, de la Salle C. Functional characterization of the human platelet glycoprotein V gene promoter: a specific marker of late megakaryocytopoietic differentiation. Blood 1999; 94: 3366-80.
    PubMedGoogle Scholar
  • 33 Ware J, Russell S, Ruggeri ZM. Generation and rescue of a murine model of platelet dysfunction – the Bernard-Soulier syndrome. Proc Natl Acad Sci USA 2000; 97: 2803-8.
    CrossrefPubMedGoogle Scholar
  • 34 Feng S, Christodoulides N, Kroll MH. The platelet glycoprotein Ib/IX complex regulates cell proliferation. Blood 1999; 93: 4256-63.
    PubMedGoogle Scholar
  • 35 Calverley DC, Kavanagh TJ, Roth GJ. Human signaling protein 14-3-3ζ interacts with platelet glycorprotein Ib subunits Ibα and Ibβ. Blood 1998; 91: 1395-403.
    PubMedGoogle Scholar
  • 36 Andrews RK, Harris SJ, McNally T, Berndt MC. Binding of purified 14-3-3 zeta signaling protein to discrete amino acid sequences within the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex. Biochemistry 1998; 37: 638-47.
    CrossrefPubMedGoogle Scholar
  • 37 Hartwig JH, DeSisto M. The cytoskeleton of the resting human blood platelet: structure of the membrane skeleton and its attachment to actin filaments. J Cell Biol 1991; 112: 407-25.
    CrossrefPubMedGoogle Scholar
  • 38 Kovacsovics TJ, Hartwig JH. Thrombin-induced GP Ib-IX centralization on the platelet surface requires actin assembly and myosin II activation. Blood 1996; 87: 618-29.
    PubMedGoogle Scholar
  • 39 Meyer SC, Zuerbig S, Cunningham CC, Hartwig JH, Bissell T, Gardner K, Fox JE. Identification of the region in actin-binding protein that binds to the cytoplasmic domain of glycoprotein Ibα. J Biol Chem 1997; 272: 2914-9.
    CrossrefPubMedGoogle Scholar
  • 40 Nurden P. Bidirectional trafficking of membrane glycoproteins following platelet activation in suspension. Thromb Haemost 1997; 78: 1305-15.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 41 Berger G, Hartwell DW, Wagner DD. P-selectin and platelet clearance. Blood 1998; 92: 4446-52.
    PubMedGoogle Scholar