Download PDF
Thromb Haemost 1988; 59(01): 054-061
DOI: 10.1055/s-0038-1642565
Review Article
Schattauer GmbH Stuttgart

Identity of Saturable Calcium-Binding Sites on Blood Platelets and Their Involvement in Platelet Aggregation

Geoffrey I Johnston
The Department of Medicine, University Hospital, Queen’s Medical Centre, Nottingham, UK
*   Current address:, Dept. of Medicine/Cardiovascular Blology Research Program, Oklahoma University Health Science Center-line 825 Northeast 13th Street Oklahoma City, Oklahoma 73104, USA
,
Stanley Heptinstall
The Department of Medicine, University Hospital, Queen’s Medical Centre, Nottingham, UK
› Author Affiliations
Further Information

Publication History

Received 07 April 1987

Accepted after revision 02 October 1987

Publication Date:
18 April 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

Extracellular Ca2+ ions are required for platelet aggregation and we show that they enter two platelet pools. One pool is rapidly filled and easily displaced by EGTA. The second is filled more slowly and is not displaced by EGTA. The EGTA-displaceable pool is believed to be surface-located and was found to contain at least one class of saturable binding sites as well as a class of non-saturable binding sites. The saturable sites were found to be highly selective for Ca2+ (dissociation constant, 3.5 × IO−7 M) even in the presence of 1 mM Mg2+ ions, and they took up between 261,000 and 307,000 Ca2+ ions/platelet. Full occupancy of the saturable binding sites appeared to be necessary for platelet aggregation to proceed. We also studied platelets that were unable to aggregate normally, either due to the congenital bleeding disorder Glanzmann’s thrombastenia or due to experimental manipulation. In both cases wc found decreased Ca2+uptake specifically by the saturable Ca2+ binding sites, and that this was associated with decreased number of GP IIb/IIIa molecules expressed on these platelets. We suggest that the Ca2+binding sites involved in platelet aggregation are located on the GP Ilb/IIIa complexes and may be involved in holding the glycoproteins in the complex together, and that the binding sites need to be fully occupied before aggregation can proceed.

Keywords

Extracellular calcium - Binding sites - Platelet aggregation
 

  • References

  • 1 Born GV R, Cross MJ. Effects of inorganic ions and of plasma proteins on the aggregation of blood platelets by ADR. J Physiol 1964; 70: 397-414
    PubMedGoogle Scholar
  • 2 Hovig T. The effect of calcium and magnesium on rabbit blood platelet aggregation in vitro. Thrombos Diathes Hemorrh 1964; 12: 179-200
    PubMedGoogle Scholar
  • 3 Mitchell JR A, Sharp AA. Platelet clumping in vitro. Br J Haematol 1964; 10: 78-93
    CrossrefPubMedGoogle Scholar
  • 4 Heptinstall S. The use of a chelating ion-exchange resin to evaluate the effects of the extracellular calcium concentration on adenosine diphosphate induced aggregation of human blood platelets. Thromb Haemostas 1976; 36: 208-220
    PubMedGoogle Scholar
  • 5 Robblee LS, Shepro D. The effect of external calcium and lanthenum on platelet calcium content and on the release reaction. Biochim Biophys Acta 1976; 436: 448-459
    CrossrefPubMedGoogle Scholar
  • 6 Peerschke EI, Grant RA, Zucker MB. Decreased association of 45calcium with platelets unable to aggregate due to thrombasthenia or prolonged calcium deprivation. Br J Haematol 1980; 46: 247-256
    CrossrefPubMedGoogle Scholar
  • 7 Brass LF, Shattil SJ. Changes in surface-bound and exchangeable calcium during platelet activation. J Biol Chem 1982; 257: 14000-14005
    PubMedGoogle Scholar
  • 8 Kunicki TJ, Pidard D, Rosa JR, Nurden AT. The formation of Ca++-dependant complexes of platelet membrane glycoproteins IIb and Ilia in solution as determined by crossed immunoelectrophoresis.. Blood 1981 58: 268-278
    PubMedGoogle Scholar
  • 9 George JN, Nurden AT, Phillips DR. Molecular defects in interactions of platelets with the vessel wall. N Engl I Med 1984; 311: 1084-1098
    CrossrefPubMedGoogle Scholar
  • 10 Brass LF, Shattil SJ. Identification and function of the high affinity binding sites for Ca2+ on the surface of platelets. J Clin Invest 1984; 73: 626-632
    CrossrefPubMedGoogle Scholar
  • 11 Peerschke EI. pH and magnesium after 45calcium binding to platelets at sites other than glycoprotein I or Ilb/IIIa. Proc Soc Exp Biol Med 1985; 179: 232-239
    CrossrefPubMedGoogle Scholar
  • 12 Zucker MB, Grant RA. Nonreversible loss of platelet aggregability induced by calcium deprivation. Blood 1978; 52: 505-514
    PubMedGoogle Scholar
  • 13 Taylor PM, Heptinstall S. The abilities of human blood platelets to bind extracellular calcium and to be aggregated by adenosine diphosphate are related. Br J Haematol 1980; 46: 115-122
    PubMedGoogle Scholar
  • 14 Fitzgerald LA, Phillips DR. Calcium regulation of the platelet membrane glycoprotein Ilb-IIIa complex. J Biol Chem 1985; 260: 11366-11374
    PubMedGoogle Scholar
  • 15 Shattil SJ, Brass LF, Bennett JS, Pandhi P. Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb—IIIa complex. Blood 1985; 66: 92-98
    PubMedGoogle Scholar
  • 16 Pidard D, Didry D, Kunicki TJ, Nurden AT. Temperature-dependent effects of EDTA on the membrane glycoprotein IIb—IIIa complex and platelet aggregability. Blood 1986; 67: 604-611
    PubMedGoogle Scholar
  • 17 Tangen O, Berman HJ. Gel filtration of blood platelets: a methodological report. Adv Exp Med Biol 1972; 34: 235-243
    PubMedGoogle Scholar
  • 18 Burgess-Wilson ME, Cockbill SR, Johnston GI, Heptinstall S. Platelet aggregation in whole blood patients with Glanzmann’s thrombasthenia. Blood 1987; 69: 38-42
    PubMedGoogle Scholar
  • 19 Portzehl H, Caldwell PC, Ruegg JC. The dependence of contraction and relaxation of muscle fibres from the crab MAIA SQUINADO on the internal concentration of free calcium ions. Biochim Biophys Acta 1964; 79: 581-591
    PubMedGoogle Scholar
  • 20 Scatchard G. The attraction of protein for small molecules and ions. Ann NY Acad Sci 1949; 51: 660-672
    CrossrefPubMedGoogle Scholar
  • 21 Adams J, Heptinstall S, Mitchell JR A. A six-channel automated platelet aggregometer. Thrombos Diathes Haemorrh 1975; 34: 821-824
    PubMedGoogle Scholar
  • 22 Johnston GI, Heptinstall S, Robins RA, Price MR. The expression of glycoproteins on single blood platelets from healthy individuals and from patients with congenital bleeding disorders. Biochem Biophys Res Comm 1984; 123: 1091-1098
    CrossrefPubMedGoogle Scholar
  • 23 Jones D, Fritschy J, Garson J, Nokes TJ C, Kemshead JT, Hardisty RM. A monoclonal antibody binding to human medulloblastoma cells and to the platelet glycoprotein IIb-IIIa complex. Br J Haematol 1984; 57: 621-631
    CrossrefPubMedGoogle Scholar
  • 24 Johnston GI, Heptinstall S. Examination of glycoproteins on intact blood platelets by using monoclonal antibodies and the fluorescence activated cell sorter. Biochem Soc Trans 1985; 13: 109 (Abstr.)
    CrossrefPubMedGoogle Scholar
  • 25 Steiner M, Tateishi T. Distribution and transport of calcium in human platelets. Biochim Biophys Acta 1974; 367: 232-246
    CrossrefPubMedGoogle Scholar
  • 26 Brass LF. Ca2+ homeostasis in unstimulated platelets. J Biol Chem 1985; 259: 12563-12570
    PubMedGoogle Scholar
  • 27 Brass LF, Shattil SJ. Identification of the saturable binding sites for Ca2+ on the surface of human platelets. Thromb Haemostas 1983; 50: 327 (Abstr.)
    PubMedGoogle Scholar
  • 28 Phillips DR, Baughan AK. Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations. J Biol Chem 1983; 258: 10240-10246
    PubMedGoogle Scholar