Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

Summary

Incorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca²⁺ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

Keywords

Platelet - Aggregation - Secretion - Calcium - Fura-2 - Quin2 - MAPT

• References

• 1 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; 94: 325-34
• 2 Grynkiewicz G, Poenie M, Tsien R Y. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-50
• 3 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; 148: 21-6
• 4 Rao G HR, Peller J D, White J G. Measurement of ionized calcium in blood platelets with a new generation calcium indicator. Biochem Biophys Res Commun 1985; 132: 652-7
• 5 Hatayama K, Kambayashi J, Nakamura K, Ohshiro T, Mori T. Fluorescent Ca²+-indicator quin2 as an intracellular Ca²+ antagonist in platelet reaction. Thromb Res 1985; 38: 505-12
• 6 Rao G HR, Peller J D, Semba C P, White J G. Influence of the calcium-sensitive fluorophore, quin2, on platelet function. Blood 1986; 67: 354-61
• 7 Giorgio T D, Hellums J D. A note on the use of quin2 in studying shear-induced platelet aggregation. Thromb Res 1986; 37: 353-9
• 8 Cazenave J P, Hemmendinger S, Beretz A, Sutter-Bay A, Launay J. L’agrégation plaquettaire: outil d’investigation clinique et d’étude pharmacologique. Méthodologie. Ann Biol Clin 1983; 41: 167-79
• 9 Costa J L, Murphy D L. Platelet 5-HT uptake and release stopped rapidly by formaldehyde. Nature 1975; 255: 407-8
• 10 Kubina M, Lanza F, Cazenave J P, Laustriat G, Kuhry J G. Parallel investigation of exocytosis kinetics and membrane fluidity changes in human platelets with the fluorescent probe, trimethylammonio-diphenylhexatriene. Biochim Biophys Acta. 1987 (in press)
• 11 Bronner C, Landry Y, Fonteneau P, Kuhry J G. A fluorescent hydrophobic probe used for monitoring the kinetics of exocytosis phenomena. Biochemistry-USA 1986; 25: 2149-54
• 12 Lanza F, Cazenave J P. Studies of alpha2-adrenergic receptors of intact and functional washed human platelets by binding of 3H-dihydroergocryptine and 3H-yohimbine. Correlation of 3H-yohimbine binding with the potentiation by adrenaline of ADP-induced aggregation Thromb Haemostas 1985; 54: 402-8
• 13 Mustard J F, Perry D W, Ardlie N G, Packham M A. Preparation of suspensions of washed platelets from humans. Brit J Haematol 1972; 22: 193-204
• 14 Tsien R Y. A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 1981; 290: 527-8
• 15 Thompson N T, Scrutton M C. Intracellular calcium fluxes in human platelets. Eur J Biochem 1985; 147: 421-7
• 16 Ware J A, Johnson P C, Smith M, Salzman E W. Effect of common agonist on cytoplasmic ionized calcium concentrations in platelets. Measurement with 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin J Clin Invest 1986; 77: 878-86
• 17 Rink T J, Pozzan T. Using quin2 in cell suspensions. Cell Calc 1985; 6: 133-44
• 18 Kinlough-Rathbone R L, Mustard J F, Packham M A, Perry P W, Reimers H J, Cazenave J P. Properties of washed human platelets. Thromb Haemostas 1977; 37: 291-308
• 19 Lupu F, Calb M, Scurei C, Simionescu N. Changes in the organization of membrane lipids during human platelet activation. Lab Invest 1986; 54: 136-45
• 20 Siegel M I, McConnell R T, Cuatrecasas P. Aspirin-like drugs interfere with arachidonate metabolism by inhibition of the 12-hydroxyperoxy-5 ,8,10,14-eicosatetraenoic acid peroxidase activity of the lipoxygenase pathway. Proc Natl Acad Sci USA 1979; 76: 3774-8
• 21 Pollock W K, Rink T J, Irvine R F. Liberation of 3H arachidonic acid and changes in cytosolic free calcium in fura-2-loaded platelets stimulated by ionomycin and collagen. Biochem J 1986; 235: 869-77