Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1984 Mar;73(3):626–632. doi: 10.1172/JCI111252

Identification and function of the high affinity binding sites for Ca2+ on the surface of platelets.

L F Brass, S J Shattil
PMCID: PMC425061  PMID: 6231306

Abstract

Extracellular Ca2+ is required for platelet aggregation and secretion in response to ADP or epinephrine. Recently, we reported that the platelet surface contains two classes of high affinity binding sites for extracellular Ca2+. To identify these sites and clarify their role in platelet function, we have now (a) studied platelets congenitally deficient in surface membrane glycoproteins and (b) examined the effect of removing surface-bound Ca2+ on platelet responses to ADP and epinephrine. Unstimulated normal platelets contained 86,000 Ca2+-binding sites/platelet with a dissociation constant (Kd) of 9 nM and 389,000 sites with a Kd of 400 nM. In contrast, thrombasthenic platelets, which lack glycoproteins IIb and IIIa, exhibited a 92% reduction in the number of higher affinity Ca2+-binding sites and a 63% reduction in the number of lower affinity sites. Bernard-Soulier platelets, which lack glycoprotein Ib, were not deficient in Ca2+-binding sites. After stimulation with ADP, both normal and thrombasthenic platelets developed approximately 138,000 new Ca2+-binding sites/platelet (Kd = 400 nM), while the larger Bernard-Soulier platelets developed 216,000 new sites. These data suggest that IIb and IIIa represent the major Ca2+-binding glycoproteins on unstimulated platelets, while neither these glycoproteins nor Ib represent the new Ca2+-binding sites on stimulated platelets. Removal of Ca2+ from the platelet surface inhibited platelet function. Despite the presence of 1 mM Mg2+, ADP- and epinephrine-induced aggregation and [14C]serotonin release were markedly decreased at free Ca2+ concentrations less than 7 nM, a value similar to the Kd of the higher affinity Ca2+-binding sites. Moreover, gadolinium, a lanthanide that competed for these Ca2+-binding sites, also inhibited aggregation and serotonin release. These studies demonstrate, therefore, that the binding of extracellular Ca2+ to glycoproteins IIb/IIIa on unstimulated platelets or to additional membrane proteins on stimulated platelets is necessary for maximal platelet responses to ADP and epinephrine. Thus, the requirement for extracellular Ca2+ during platelet activation by these agonists may actually represent a requirement for surface-bound Ca2+.

Full text

PDF
626

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bennett J. S., Hoxie J. A., Leitman S. F., Vilaire G., Cines D. B. Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody. Proc Natl Acad Sci U S A. 1983 May;80(9):2417–2421. doi: 10.1073/pnas.80.9.2417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett J. S., Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest. 1979 Nov;64(5):1393–1401. doi: 10.1172/JCI109597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bers D. M. A simple method for the accurate determination of free [Ca] in Ca-EGTA solutions. Am J Physiol. 1982 May;242(5):C404–C408. doi: 10.1152/ajpcell.1982.242.5.C404. [DOI] [PubMed] [Google Scholar]
  4. Best L. C., Bone E. A., Jones P. B., Russell R. G. Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets. Biochim Biophys Acta. 1980 Oct 1;632(2):336–342. doi: 10.1016/0304-4165(80)90091-4. [DOI] [PubMed] [Google Scholar]
  5. Brass L. F., Shattil S. J. Changes in surface-bound and exchangeable calcium during platelet activation. J Biol Chem. 1982 Dec 10;257(23):14000–14005. [PubMed] [Google Scholar]
  6. Clemetson K. J., McGregor J. L., James E., Dechavanne M., Lüscher E. F. Characterization of the platelet membrane glycoprotein abnormalities in Bernard-Soulier syndrome and comparison with normal by surface-labeling techniques and high-resolution two-dimensional gel electrophoresis. J Clin Invest. 1982 Aug;70(2):304–311. doi: 10.1172/JCI110618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Marco L., Shapiro S. S. Properties of human asialo-factor VIII. A ristocetin-independent platelet-aggregating agent. J Clin Invest. 1981 Aug;68(2):321–328. doi: 10.1172/JCI110259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feinstein M. B. The role of calmodulin in hemostasis. Prog Hemost Thromb. 1982;6:25–61. [PubMed] [Google Scholar]
  9. Howard L., Shulman S., Sadanandan S., Karpatkin S. Crossed immunoelectrophoresis of human platelet membranes. The major antigen consists of a complex of glycoproteins, GPIIb and GPIIIa, held together by Ca2+ and missing in Glanzmann's thrombasthenia. J Biol Chem. 1982 Jul 25;257(14):8331–8336. [PubMed] [Google Scholar]
  10. Jennings L. K., Phillips D. R. Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-III complex. J Biol Chem. 1982 Sep 10;257(17):10458–10466. [PubMed] [Google Scholar]
  11. Kaibuchi K., Sano K., Hoshijima M., Takai Y., Nishizuka Y. Phosphatidylinositol turnover in platelet activation; calcium mobilization and protein phosphorylation. Cell Calcium. 1982 Oct;3(4-5):323–335. doi: 10.1016/0143-4160(82)90020-3. [DOI] [PubMed] [Google Scholar]
  12. Kretsinger R. H. The informational role of calcium in the cytosol. Adv Cyclic Nucleotide Res. 1979;11:1–26. [PubMed] [Google Scholar]
  13. Lages B., Weiss H. J. Dependence of human platelet functional responses on divalent cations: aggregation and secretion in heparin- and hirudin-anticoagulated platelet-rich plasma and the effects of chelating agents. Thromb Haemost. 1981 Apr 30;45(2):173–179. [PubMed] [Google Scholar]
  14. McGregor J. L., Clemetson K. J., James E., Capitanio A., Greenland T., Lüscher E. F., Dechavanne M. Glycoproteins of platelet membranes from Glanzmann's thrombasthenia. A comparison with normal using carbohydrate-specific or protein-specific labelling techniques and high-resolution two-dimensional gel electrophoresis. Eur J Biochem. 1981 May 15;116(2):379–388. doi: 10.1111/j.1432-1033.1981.tb05346.x. [DOI] [PubMed] [Google Scholar]
  15. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  16. PORTZEHL H., CALDWELL P. C., RUEEGG J. C. 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 May 25;79:581–591. doi: 10.1016/0926-6577(64)90224-4. [DOI] [PubMed] [Google Scholar]
  17. Peerschke E. I., Grant R. A., Zucker M. B. Decreased association of 45calcium with platelets unable to aggregate due to thrombasthenia or prolonged calcium deprivation. Br J Haematol. 1980 Oct;46(2):247–256. doi: 10.1111/j.1365-2141.1980.tb05963.x. [DOI] [PubMed] [Google Scholar]
  18. Peerschke E. I., Zucker M. B., Grant R. A., Egan J. J., Johnson M. M. Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood. 1980 May;55(5):841–847. [PubMed] [Google Scholar]
  19. Peterson D. M., Wehring B. Isoelectric characteristics and surface radioiodination of normal and thrombasthenic platelet membrane glycoproteins. Thromb Res. 1981 Apr 1;22(1-2):53–65. doi: 10.1016/0049-3848(81)90308-x. [DOI] [PubMed] [Google Scholar]
  20. Scrutton M. C., Egan C. M. Divalent cation requirements for aggregation of human blood platelets and the role of the anti-coagulant. Thromb Res. 1979;14(4-5):713–727. doi: 10.1016/0049-3848(79)90127-0. [DOI] [PubMed] [Google Scholar]
  21. Shattil S. J., Montgomery J. A., Chiang P. K. The effect of pharmacologic inhibition of phospholipid methylation on human platelet function. Blood. 1982 May;59(5):906–912. [PubMed] [Google Scholar]
  22. Tangen O., Berman H. J., Marfey P. Gel filtration. A new technique for separation of blood platelets from plasma. Thromb Diath Haemorrh. 1971 Jun 30;25(2):268–278. [PubMed] [Google Scholar]
  23. Walsh P. N., Mills D. C., Pareti F. I., Stewart G. J., Macfarlane D. E., Johnson M. M., Egan J. J. Hereditary giant platelet syndrome. Absence of collagen-induced coagulant activity and deficiency of factor-XI binding to platelets. Br J Haematol. 1975 Apr;29(4):639–655. doi: 10.1111/j.1365-2141.1975.tb02750.x. [DOI] [PubMed] [Google Scholar]
  24. Zucker M. B., Grant R. A. Nonreversible loss of platelet aggregability induced by calcium deprivation. Blood. 1978 Sep;52(3):505–513. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES