Download PDF
Thromb Haemost 1997; 78(06): 1510-1515
DOI: 10.1055/s-0038-1665443
Rapid Communication
Schattauer GmbH Stuttgart

Peptide-specific Antibodies as Pro\bes of the Topography of the Ca2+-binding Motifs in αIIbβ3 [*]

Jolanta Niewiarowska
1   The Department of Biophysics, Medical University in Lodz, Poland
,
Mariola Świderska
1   The Department of Biophysics, Medical University in Lodz, Poland
,
Tadeusz Majewski
2   Department of Chemistry, University of Warsaw, Poland
,
Czesław S Cierniewski
1   The Department of Biophysics, Medical University in Lodz, Poland
3   Center for Microbiology and Virology, Poland
4   Polish Academy of Sciences, Lodz, Poland
› Author Affiliations
Further Information

Publication History

Received 05 1997

Accepted after resubmission 04 August 1997

Publication Date:
12 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

Antibodies against synthetic peptides corresponding to four Ca2+- binding motifs of the αIIb subunit have been obtained and used as molecular probes to analyze the topography of the αIIbβ3 complex. The specificity of the antibodies has been characterized by ELISA and Western immunoblotting in terms of binding capacity and affinity to the isolated αIIbβ3 and its αIIb subunit. Our data suggest that: (a) all four Ca2+-binding motifs of the αIIb are partially exposed on the surface of the intact molecule and accessible to antipeptide antibodies. However, they are not in close vicinity to the ligand recognition domain since the antibodies do not produce complete inhibition of platelet aggregation, (b) The conformation of amino acid stretches which form the second Ca2+-binding motif of αIIb is particularly dependent upon the presence of cation, and this region undergoes significant conformational alterations upon Ca2+ expulsion.

*This work was supported in part by the grant PAN/HHS-96-242 from the US-Polish Maria Sklodowska-Curie Joint Fund II, the grant 15195-543001 from Howard Hughes Medical Institute, and PECO project ERBCIPDCT 940247


 

  • References

  • 1 Hynes RO. Integrins: A family of cell surface receptors. Cell 1987; 48: 549-554
    CrossrefPubMedGoogle Scholar
  • 2 Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987; 238: 491-497
    CrossrefPubMedGoogle Scholar
  • 3 Ginsberg MH, Loftus JC, Plow EF. Cytoadhesins, integrins and platelets. Thromb Haemost 1988; 59: 1-6
    Article in Thieme ConnectPubMedGoogle Scholar
  • 4 Akiyama SK, Nagata K, Yamada KM. All surface receptors for extracellular matrix components. Biochim Biophys Acta 1990; 1031: 091-110
    CrossrefPubMedGoogle Scholar
  • 5 Springer TA. Adhesion receptors of the immune system. Nature (London) 1990; 346: 425-434
    CrossrefPubMedGoogle Scholar
  • 6 Gailit J, Ruoslahti E. Regulation of the fibronectin receptor affinity by divalent cations. J Biol Chem 1988; 263: 12927-12932
    PubMedGoogle Scholar
  • 7 Kirchhofer D, Grzesiak J, Pierschbacher MD. Calcium as a potential physiological regulator of integrin-mediated cell adhesion. J Biol Chem 1991; 266: 4471-4477
    PubMedGoogle Scholar
  • 8 Smith JW, Cheresh DA. Labeling of integrin αIIbβ3 with 58Co(III). J Biol Chem 1991; 266: 11429-11432
    PubMedGoogle Scholar
  • 9 D’Souza SE, Haas TA, Piotrowicz RS, Byers-Ward V, McGrath DE, Soule HR, Ciemiewski CS, Plow EF, Smith JW. Ligand and cation binding are dual function of a discrete segment of the integrin (β3 subunit: Cation displacement is involved in ligand binding. Cell 1994; 79: 659-667
    CrossrefPubMedGoogle Scholar
  • 10 Strynadka N, James M. Crystal structures of the helix-loop-helix calcium binding proteins. Ann Rev Biochem 1989; 58: 951-998
    CrossrefPubMedGoogle Scholar
  • 11 Tuckwell DS, Brass A, Humphries MJ. Homology modelling of integrin EF-hands. Biochem J 1992; 285: 325-331
    CrossrefPubMedGoogle Scholar
  • 12 Gulino D, Boudignon C, Zhang LY, Concord E, Rabiet MJ, Marguerie G. Ca2+-binding properties of the platelet glycoprotein IIb ligand-interacting domain. J Biol Chem 1992; 267: 1001-1007
    PubMedGoogle Scholar
  • 13 Ciemiewski CS, Haas TA, Smith JW, Plow EF. Characterization of cation-binding sequences in the platelet integrin GPIIb-IIIa (αIIbβ3) by terbium luminescence. Biochemistry 1994; 33: 12238-12246
    CrossrefPubMedGoogle Scholar
  • 14 Basani RB, Vilaire G, Shattil SJ, Kolodziej MA, Bennett JS, Poncz M. Glanzmann Thrombasthenia due to a two amino acid deletion in the fourth calcium-binding domain of alpha IIb: demonstration of the importance of calcium-binding domain in the conformation of alpha IIb beta 3. Blood 1996; 88: 167-173
    PubMedGoogle Scholar
  • 15 Pytela R, Pierschbacher MD, Ginsberg MH, Plow EF, Ruoslahti E. Platelet membrane glycoprotein IIb/IIIa: Member of a family of Arg-Gly-Asp-specific adhesion receptors. Science 1986; 231: 1559-1562
    CrossrefPubMedGoogle Scholar
  • 16 Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedGoogle Scholar
  • 17 Doolittle RF, Schubert D, Schwartz SA. Amino acid sequence studies on artiodactyl fibrinopeptides I. Dromadery camel mule deer and cape buffalo. Arch Biochem Biophys 1967; 118: 456-465
    CrossrefPubMedGoogle Scholar
  • 18 Merrifield RB. Solid phase peptide synthesis. I. The synthesis of tetrapep-tide. J Am Chem Soc 1963; 85: 2149-2154
    CrossrefPubMedGoogle Scholar
  • 19 Mitchell AR, Kent SBH, Engelhard M, Merrifield RB. A new synthetic route to tert-butyloxycarbonylaminoacyl-4-(oxymethyl)phenylacetami-domethyl-resin, an improved support for solid-phase peptide synthesis. J Org Chem 1978; 43: 2845-2852
    CrossrefPubMedGoogle Scholar
  • 20 Manning M, Wuu TC, Baxter JWM. The purification of synthetic oxytocin and analogues by gel filtration on Sephadex G-15. J Chromatogr 1968; 38: 396-398
    CrossrefPubMedGoogle Scholar
  • 21 Avrameas S, Temyck T. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 1969; 6: 53-66
    CrossrefPubMedGoogle Scholar
  • 22 Stocker JW, Malavasis F, Trucco M. In: Immunological methods. Lefkovits I, Benvenuto P. eds Academic Press London; New York San Francisco: 1981. 02. pp 299-308
    Crossref
  • 23 Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with staining. J Biol Chem 1951; 193: 265-275
    PubMedGoogle Scholar
  • 24 Towbin H, Staehelin T, Gordon J. Electrophoretic from poliacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354
    CrossrefPubMedGoogle Scholar
  • 25 Zar J. Biostatistical Analysis. Prentice-Hall. Englewood Clifs; NJ: 1984
    Google Scholar
  • 26 Mustard JF, Perry DW, Ardlie NC, Paccham MA. Preparation of suspensions of washed platelet from humans. Brit J Haematol 1977; 22: 193-204
    PubMedGoogle Scholar
  • 27 Niewiarowska J, Ciemiewski CS. GPIIIa(90-102) and GPIIIa(631-653) epitopes as markers of conformational changes occuring during the activation of glycoprotein IIb/IIIa complex. Eur J Biochem 1994; 224: 8039
    PubMedGoogle Scholar