SPATIAL-DISTRIBUTION AND TEMPORAL CHANGE IN CYTOSOLIC PH AND [CA2+] IN RESTING AND ACTIVATED SINGLE HUMAN PLATELETS

In human platelets, a rapid rise in cytoplasmic Ca2+ and slower rise in cytoplasmic pH follow stimulation by thrombin. With the Ca2+ probe Fura-2 and the pH probe SNARF-1 for digitized fluorescence microscopy, we studied simultaneously the distribution and changes with time of [pH](i) and [Ca2+](i) in individual human platelets. In platelets co-loaded with both probes, the probes had no detectable fluorescence at each other's excitation wavelength. The monovalent cation ionophore, nigericin (2 mu M), produced a homogeneous rise in pH but no change in [Ca2+]. Platelets, in contact with glass, spread and developed an irregular, apparently mutually independent rise in both [Ca2+] and pH. Stimulation of platelets by thrombin 1.0 U/ml elevated [Ca2+](i) and produced slow alkalinization without initial acidification. Replacement of extracellular Na+ by choline abolished thrombin-induced alkalinization, but had no effect on thrombin-induced [Ca2+](i) elevation. ADP 10 mu M caused a rapid rise of [Ca2+](i) and transient alkalinization. Most stimulated platelets developed a gradient in pH, that was highest in the center. ADP and thrombin caused oscillation of [Ca2+](i) but not of [pH](i). We conclude that alkalinization in stimulated platelets, presumably involving Na+/H+ antiport, is not essential for the rise of [Ca2+](i) that may accompany it.