[Significance of multiple analysis of platelet Ca2+ mobilization by using some available methods measuring cytosolic Ca2+ concentration, membrane-bound Ca2+ and Ca2+ flux across the plasma membrane].
Intracellular Ca2+ mobilization in the resting platelets undergoing Ca2+ influx and in the activated platelets stimulated by thrombin and phorbol ester (TPA) was investigated by measuring cytosolic Ca2+ concentration: [Ca2+]cyt (fura-2, aequorin), membrane-bound Ca2+ (chlortetracycline) and Ca2+ flux across the plasma membrane (45Ca2+ tracer). Some modified methods employed in this study concerning 45Ca2+ flux measurement and aequorin loading into platelets were very useful to elucidate the details of platelet Ca2+ mobilization in the combination with other Ca2+ assay methods. These combined and integrated assays on platelet Ca2+ showed some new interesting findings as follows; (1) The difference of the obtained values of [Ca2+]cyt and of the reaction pattern between fura-2 and aequorin method was responsible to the difference of the distribution of probe molecules in platelet. This was confirmed by the higher sensitivity of aequorin molecules to [Ca2+]cyt elevation induced by Ca2+ influx than by that of fura-2 molecules in thrombin- and TPA-activated platelets. (2) The membrane-bound Ca2+ release in activated platelets was found to be another intracellular Ca2+ movement distinct from organelle Ca2+ release and CTC molecules not to bind to the organelle membranes. (3) The plasma membrane Ca2+ pump was found to exist in both unstimulated platelets and stimulated platelets suggested by its specific time course of 45Ca2+ binding and [Ca2+]cyt increase in platelets. (4) There existed biphasic increase of [Ca2+]cyt in thrombin-stimulated platelets consisted of biphasic Ca2+ influx and biphasic intracellular Ca2+ release with alternating point at 10 seconds after activation. The initial changes seemed to be correlated with receptor operated Ca2+ channel and receptor-linked phospholipase C activation respectively, while both second phase increase seemed to be correlated with the plasma membrane phospholipid metabolisms evoked by phospholipase A2 activation.
Department of Laboratory Medicine, Hokkaido University School of Medicine, Sapporo.
F
F
Katabami
D000331:Aequorin; D001572:Benzofurans; D001792:Blood Platelets / Q000032:analysis*; D002118:Calcium / Q000097:blood*; D002462:Cell Membrane / Q000378:metabolism; D002751:Chlortetracycline; D003600:Cytosol / Q000378:metabolism*; D016257:Fura-2; D006801:Humans; D010974:Platelet Aggregation; D013050:Spectrometry, Fluorescence / Q000379:methods
D001572:Benzofurans; D000331:Aequorin; D002118:Calcium; D016257:Fura-2; D002751:Chlortetracycline