Selective inhibition of human platelet phospholipase A 2 by buffering cytoplasmic calcium with the fluorescent indicator quin 2. Evidence for different calcium sensitivities of phospholipases A 2 and C

Abstract

Human platelets labelled with either [ 14C]arachidonic acid or [ 32P]orthophosphate were loaded or not with the Ca 2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1U/ml) in a medium where Ca 2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A 2 activity, as detected by the release of [ 14C]arachidonate and of its metabolites, or by the hydrolysis of [ 14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca 2+. However, Ca 2+ was not required in non-loaded platelets, where a maximal phospholipase A 2 activity was detected in the absence of external Ca 2+. In contrast, phospholipase C action, as determined from the amounts of [ 14C]diacylglycerol, [ 14C]- or [ 32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca 2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A 2 could be overcome by external Ca 2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A 2 activity was fully suppressed even in the presence of external Ca 2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A 2 requires higher Ca 2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A 2 activity under various conditions with the values of cytoplasmic free Ca 2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca 2+ in control platelets stimulated with thrombin can attain concentrations above 1 μM, probably close to 5–10 μM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069μ-2076).