Simultaneous flow cytometric measurements of thrombin-induced cytosolic pH and Ca2+ fluxes in human platelets.
Human platelets exhibit an extremely rapid increase in cytoplasmic Ca2+ concentrations ((Ca2+]in) and a dose-dependent cytoplasmic pH change ((pH]in) upon thrombin stimulation. A cytoplasmic alkalinization, maximal by 60 s, is preceded by a very rapid acidification, which is masked by the alkalinization when saturating thrombin doses are used. Using the pH probe 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein we report here the kinetics of simultaneous cytoplasmic pH and Ca2+ changes in thrombin-stimulated platelets, measured in single cells by flow cytometry. This permits analysis of the responding subpopulation. Maximal thrombin stimulation (greater than or equal to 4.5 nM) induces a dose-dependent increase in pHin from approximately 7.0 to 7.30 and a maximal [Ca2+]in transient of up to 800 nM. The Ca2+ transient coincides temporally with the rapid initial acidification, while the alkalinization is maximal considerably later. The Ca2+ transients occur maximally in each responding cell, but occur only in a subpopulation of the platelets at subsaturating (less than 4.5 nM) thrombin doses; in contrast, the dose-dependent cytoplasmic acidification appears to occur uniformly in all platelets. The rapid increase in [Ca2+]in is not dependent on the alkalinization, and the former occurs maximally in amiloride treated, Na+/H+ exchange inhibited human platelets. These results indicate that the acidification and the rise in [Ca2+]in may be interrelated, whereas the cytoplasmic alkalinization (maximal considerably later than either the acidification or the [Ca2+]in rise) may be independent of these earlier, temporally correlated increases in H+ and Ca2+ concentrations.
Davies
T A
TA
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
Weil
G J
GJ
Simons
E R
ER
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D003600:Cytosol / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D013917:Thrombin / Q000494:pharmacology*
D005456:Fluorescent Dyes; D013917:Thrombin; D002118:Calcium