Thromb Haemost 1992; 68(03): 352-356
DOI: 10.1055/s-0038-1656379
Original Article
Schattauer GmbH Stuttgart

On the Significance of Different Aequorin Loading Techniques on Intracellular Aequorin Discharge, Baseline Calcium, Platelet Aggregation and Aequorin-Indicated Ca2+-Transients

R Malmgren
1   The Department of Experimental Surgery, Karolinska Institute, Stockholm, Sweden
,
S Grunfelt
2   The Department of Neurology, Henry Fords Hospital, Detroit, Ml, USA
,
Rajiv Joseph
2   The Department of Neurology, Henry Fords Hospital, Detroit, Ml, USA
› Institutsangaben
Weitere Informationen

Publikationsverlauf

Received 06. November 1991

Accepted after revision 16. April 1992

Publikationsdatum:
04. Juli 2018 (online)

Summary

The study compares the decay of intracellular luminescence activity (L max), the levels of basal [Ca2+]i in resting platelets, and agonist-induced peak [Ca2+]i-signals in platelets loaded with aequorin using the EGTA-, DMSO- and hypoosmotic shock treatment (HOST)-techniques. The highest load of intracellular aequorin with almost unchanged luminescence activity during 4 h was achieved with HOST. L max decreased linearly in EGTA- and HOST-platelets, but the decay rate and the levels of basal [Ca2+]i were significantly lower in HOST-platelets. Platelet aggregation and aequorin-indicated [Ca2+]i-rise induced by thrombin and collagen were similar in EGTA- and HOST-platelets. In HOST-platelets, ADP-induced platelet aggregation was always accompanied by aequorin-signals, while at a similar time point, aequorin-signals were absent in 3 of 5 cases in EGTA-platelets. The initial aequorin loading was highest in DMSO-platelets, but L max described an exponential decay, which was most pronounced when DMSO-platelets were maintained in Ca2+-free buffer (R 2 = 0.86). Agonist-induced platelet aggregation was significantly reduced in DMSO-platelets: thrombin-stimulation was accompanied by a significantly lower and delayed [Ca2+]i-rise and no aequorin-signal was obtained in response to ADP in 3 of 5 cases. The study shows that in addition of being a rapid loading-technique, the criteria of high intracellular aequorin load with low luminescence consumption, low basal [Ca2+]i and completely preserved platelet functions are most convincingly met by the HOST-method.

 
  • References

  • 1 Snowdowne KW, Borle AB. Measurements of cytosolic free calcium in mammalian cells with aequorin. Am J Physiol 1984; 247: C396-C408
  • 2 Johnson PC, Ware JA, Cliveden PB, Smith M, Dvorak AM, Salzman EW. Measurement of ionized calcium in blood platelets with the photoprotein aequorin. J Biol Chem 1985; 260: 2069-2076
  • 3 Blinks JR. Use of calcium-regulated photoproteins as intracellular Ca2+ indicators. Methods Enzymol 1989; 172: 164-203
  • 4 Johnson PC, Ware JA, Salzman EW. Concurrent measurement of platelet ionized calcium concentration and aggregation: studies with the lumiaggregometer. Thromb Res 1985; 40: 435-443
  • 5 Yamaguchi A, Suzuki H, Tanoue K, Yamasaki H. Simple method of aequorin loading into platelets using dimethyl sulfoxide. Thromb Res 1986; 44: 165-174
  • 6 Vickers JD, Mustard JF. The phosphoinositides exist in multiple metablic pools in rabbit platelets. Biochem J 1986; 238: 411-417
  • 7 Steen MV. Human platelet signal transduction and responses: A study with special reference to the platelet-stimulating effect of epinephrine. Dissertation, University of Bergen: Bergen, Norway: 1990
  • 8 Rink TJ, Tsien RY, Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol 1982; 95: 189-196
  • 9 Hess P, Meetzger P, Weingart R. Free magnesium in sheep, ferret and frog striated muscle at rest measured with ion-selective microelectrodes. J Physiol 1982; 333: 173-188
  • 10 Ware JA, Smith M, Fossel ET, Salzman EW. Cytoplasmic Mg2+ concentration in platelets: implications for determination of Ca2+ with aequorin. Am J Physiol 1988; 255: H855-H859
  • 11 Benett WF, Lynch G. Low-temperature induction of calcium-dependent protein phosphorylation in blood platelets. J Cell Biol 1980; 86: 280-285
  • 12 Pribluda V, Rotman A. Dynamics of membrane-cytoskeleton interaction in activated blood platelets. Biochemistry 1982; 21: 2825-2832
  • 13 Lages B, Weiss JH. Time dependence of aequorin-indicated calcium levels in stimulated and unstimulated platelets: Evidence for multiple aequorin environments in platelets. Thromb Haemostas 1989; 62: 1094-1099
  • 14 Ware JA, Johnson PC, Smith M, Salzman EW. Effect of common agonists on cytoplasmic ionized calcium concentration in platelets. J Clin Invest 1986; 77: 878-886
  • 15 Potevin F, Lecompte T, Favier R, Samama M. Rapid aequorin loading into platelets in the presence of DMSO – characteristics of the responses (changes in light transmission and in calcium) to various agonists. Thromb Haemostas 1991; 66: 334-342
  • 16 Joseph Rajiv, Han E, Grunfelt S, Welch KMA. Dimethyl sulfoxide and stimulation of platelet cytosolic calcium. Thromb Haemostas 1989; 61: 326
  • 17 Lehuu B, Curtis-Prior PB. Effect of dimethyl sulfoxide (DMSO) on aggregation of human blood platelets. J Pharm Pharmacol 1987; 39: 62-63
  • 18 Nakano T, Terawaki A, Arita H. Measurement of thromboxane A2-induced elevation of ionized calcium in collagen-stimulated platelets with the photoprotein aequorin. J Biochem 1986; 99: 1285-1288