Skip to main content
The Journal of Physiology logoLink to The Journal of Physiology
. 1985 Nov;368:131–146. doi: 10.1113/jphysiol.1985.sp015850

Responses to adenosine diphosphate in human platelets loaded with the fluorescent calcium indicator quin2.

T J Hallam, T J Rink
PMCID: PMC1192589  PMID: 4078739

Abstract

ADP produces a rapid elevation in the concentration of cytoplasmic free calcium, [Ca2+]i, in quin2-loaded human platelets which begins within 1 s of stimulation and peaks after 10 s. In the presence of 1 mM-extracellular calcium, [Ca2+]i peaks at 670 +/- 50 nM in the absence and 610 +/- 30 nM in the presence of a cyclo-oxygenase inhibitor. The production of prostaglandin endoperoxides and thromboxane A2 are not required for stimulation of Ca2+ fluxes by ADP but appear to have a supportive role. In the absence of extracellular calcium ions and with 1 mM-extracellular EGTA, stimulation with ADP caused [Ca2+]i to peak at 160 +/- 20 nM in the absence and 150 +/- 10 nM in the presence of a cyclo-oxygenase inhibitor. ADP can cause the discharge of calcium ions from internal stores and does not require the prior formation of prostaglandin endoperoxides or thromboxane A2. The rise in [Ca2+]i in the presence of extracellular Ca2+ is sixfold larger than in the absence of extracellular Ca2+. This suggests that the major component of the ADP-stimulated rise in [Ca2+]i is caused by the influx of Ca2+ ions across the plasma membrane. Diltiazem, D600, nimodipine and nifedipine had little or no effect on resting or ADP-stimulated [Ca2+]i levels. Depolarization with potassium-rich media alone or in conjunction with valinomycin had no effect on basal [Ca2+]i and only a partial inhibitory effect on ADP-stimulated increases in [Ca2+]i. Depolarization had no effect on the ADP-stimulated rise in [Ca2+]i in Ca2+-free media. Hyperpolarization had no marked effect on the rise in [Ca2+]i produced by ADP in the presence of extracellular calcium. These results are consistent with there being no voltage-dependent channels in the platelet plasma membrane. Using ionomycin, a selective Ca2+ ionophore, and measuring both quin2 fluorescence and optical density of the suspension simultaneously, the threshold [Ca2+]i for shape change was determined to be 300 nM with half-maximal effect at 500 nM and maximal shape change at 800 nM. ADP produced maximal shape change confirmed by scanning electron microscopy with corresponding [Ca2+]i at below 200 nM. The level of [Ca2+]i required to produce aggregation using ionomycin was approximately 1 microM. ADP alone, or following a smaller rise in [Ca2+]i produced by ionomycin to disguise the effect of ADP, produced an aggregatory response at concentrations below 1 microM. These data indicate that excitatory mechanisms are involved producing shape change and aggregation to ADP other than a stimulated rise in [Ca2+]i.

Full text

PDF
131

Images in this article

Selected References

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

  1. BORN G. V. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. 1962 Jun 9;194:927–929. doi: 10.1038/194927b0. [DOI] [PubMed] [Google Scholar]
  2. Baker P. F., Rink T. J. Catecholamine release from bovine adrenal medulla in response to maintained depolarization. J Physiol. 1975 Dec;253(2):593–620. doi: 10.1113/jphysiol.1975.sp011209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  5. Best L. C., Holland T. K., Jones P. B., Russell R. G. The interrelationship between thromboxane biosynthesis, aggregation and 5-hydroxytryptamine secretion in human platelets in vitro. Thromb Haemost. 1980 Feb 29;43(1):38–40. [PubMed] [Google Scholar]
  6. Born G. V. Fluid-mechanical and biochemical interactions in haemostasis. Br Med Bull. 1977 Sep;33(3):193–197. doi: 10.1093/oxfordjournals.bmb.a071435. [DOI] [PubMed] [Google Scholar]
  7. Born G. V. Observations on the change in shape of blood platelets brought about by adenosine diphosphate. J Physiol. 1970 Aug;209(2):487–511. doi: 10.1113/jphysiol.1970.sp009176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Feinstein M. B., Fraser C. Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP. J Gen Physiol. 1975 Nov;66(5):561–581. doi: 10.1085/jgp.66.5.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hallam T. J., Sanchez A., Rink T. J. Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2. Biochem J. 1984 Mar 15;218(3):819–827. doi: 10.1042/bj2180819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hallam T. J., Thompson N. T., Scrutton M. C., Rink T. J. The role of cytoplasmic free calcium in the responses of quin2-loaded human platelets to vasopressin. Biochem J. 1984 Aug 1;221(3):897–901. doi: 10.1042/bj2210897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jones D., Fritschy J., Garson J., Nokes T. J., Kemshead J. T., Hardisty R. M. A monoclonal antibody binding to human medulloblastoma cells and to the platelet glycoprotein IIB-IIIA complex. Br J Haematol. 1984 Aug;57(4):621–631. doi: 10.1111/j.1365-2141.1984.tb02939.x. [DOI] [PubMed] [Google Scholar]
  13. Lee T. C., Malone B., Blank M. L., Snyder F. 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) stimulates calcium influx in rabbit platelets. Biochem Biophys Res Commun. 1981 Oct 30;102(4):1262–1268. doi: 10.1016/s0006-291x(81)80147-7. [DOI] [PubMed] [Google Scholar]
  14. Lloyd J. V., Nishizawa E. E., Mustard J. F. Effect of ADP-induced shape change on incorporation of 32P into platelet phosphatidic acid and mono-, di- and triphosphatidyl inositol. Br J Haematol. 1973 Jul;25(1):77–99. doi: 10.1111/j.1365-2141.1973.tb01718.x. [DOI] [PubMed] [Google Scholar]
  15. MacIntyre D. E., Rink T. J. The role of platelet membrane potential in the initiation of platelet aggregation. Thromb Haemost. 1982 Feb 26;47(1):22–26. [PubMed] [Google Scholar]
  16. MacIntyre D. E., Shaw A. M. Phospholipid-induced human platelet activation: effects of calcium channel blockers and calcium chelators. Thromb Res. 1983 Sep 15;31(6):833–844. doi: 10.1016/0049-3848(83)90114-7. [DOI] [PubMed] [Google Scholar]
  17. Massini P., Lüscher E. F. On the significance of the influx of calcium ions into stimulated human blood platelets. Biochim Biophys Acta. 1976 Jul 1;436(3):652–663. doi: 10.1016/0005-2736(76)90447-8. [DOI] [PubMed] [Google Scholar]
  18. Motulsky H. J., Snavely M. D., Hughes R. J., Insel P. A. Interaction of verapamil and other calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors. Circ Res. 1983 Feb;52(2):226–231. doi: 10.1161/01.res.52.2.226. [DOI] [PubMed] [Google Scholar]
  19. Mustard J. F., Perry D. W., Ardlie N. G., Packham M. A. Preparation of suspensions of washed platelets from humans. Br J Haematol. 1972 Feb;22(2):193–204. doi: 10.1111/j.1365-2141.1972.tb08800.x. [DOI] [PubMed] [Google Scholar]
  20. Mustard J. F., Perry D. W., Kinlough-Rathbone R. L., Packham M. A. Factors responsible for ADP-induced release reaction of human platelets. Am J Physiol. 1975 Jun;228(6):1757–1765. doi: 10.1152/ajplegacy.1975.228.6.1757. [DOI] [PubMed] [Google Scholar]
  21. O'Rourke F. A., Halenda S. P., Zavoico G. B., Feinstein M. B. Inositol 1,4,5-trisphosphate releases Ca2+ from a Ca2+-transporting membrane vesicle fraction derived from human platelets. J Biol Chem. 1985 Jan 25;260(2):956–962. [PubMed] [Google Scholar]
  22. Owen N. E., Feinberg H., Le Breton G. C. Epinephrine induces Ca2+ uptake in human blood platelets. Am J Physiol. 1980 Oct;239(4):H483–H488. doi: 10.1152/ajpheart.1980.239.4.H483. [DOI] [PubMed] [Google Scholar]
  23. Phillips D. R., Baughan A. K. Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations. J Biol Chem. 1983 Sep 10;258(17):10240–10246. [PubMed] [Google Scholar]
  24. Rink R. J., Sanchez A., Grinstein S., Rothstein A. Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca2+ concentration. Biochim Biophys Acta. 1983 Jul 14;762(4):593–596. doi: 10.1016/0167-4889(83)90064-2. [DOI] [PubMed] [Google Scholar]
  25. Rink T. J., Sanchez A., Hallam T. J. Diacylglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets. Nature. 1983 Sep 22;305(5932):317–319. doi: 10.1038/305317a0. [DOI] [PubMed] [Google Scholar]
  26. Rink T. J., Smith S. W., Tsien R. Y. Cytoplasmic free Ca2+ in human platelets: Ca2+ thresholds and Ca-independent activation for shape-change and secretion. FEBS Lett. 1982 Nov 1;148(1):21–26. doi: 10.1016/0014-5793(82)81234-9. [DOI] [PubMed] [Google Scholar]
  27. Tsien R. W. Calcium channels in excitable cell membranes. Annu Rev Physiol. 1983;45:341–358. doi: 10.1146/annurev.ph.45.030183.002013. [DOI] [PubMed] [Google Scholar]
  28. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. White J. G. Current concepts of platelet structure. Am J Clin Pathol. 1979 Apr;71(4):363–378. doi: 10.1093/ajcp/71.4.363. [DOI] [PubMed] [Google Scholar]
  30. White J. G., Rao G. H., Gerrard J. M. Effects of the lonophore A23187 on blood platelets I. Influence on aggregation and secretion. Am J Pathol. 1974 Nov;77(2):135–149. [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES