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.

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)-S(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 (~4.5 nM) induces a dosedependent increase in pHi" 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 (~4.5 nM) thrombin doses; in contrast, the dose-dependent cytoplasmic acidification appears to occur uniformly in all platelets. The rapid increase in [Ca%, 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 [Ca"']i, rise) may be independent of these earlier, temporally correlated increases in H+ and Ca2' concentrations.
We have previously shown that stimulation of human platelets by cu-thrombin is accompanied by a rapid dose-dependent membrane depolarization (l-3) and a cytoplasmic alkalinization (4-7). These changes start immediately, but attain their maxima later than the temporally correlated rapid Ca*+ transient (8)(9)(10)(11) and the formation of products of phosphoinositide metabolism (11)(12)(13)(14)(15)(16)(17)(18)(19)(20); however, they precede platelet lysosomal granule release (6) and aggregation (20). While the role of the cytoplasmic alkalinization in the overall mechanism of platelet activation is unclear, it appears to be coupled to a concomitant sodium influx since both are blocked with dimethyl amiloride (3,5,21,23). The acidification is not abolished by blockage of Na+/H' countertransport nor by chelation of cytoplasmic Ca*+ after amiloride (21) and BAPTA (5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-te-* (30). The calibration curve utilizing the ratio technique ( Fig. la) indicated that the resting pH of the platelet (the pH at which nigericin yields no change in fluorescence) was 6.97 + 0.04 (n = 26, mean k SD.); a comparable curve using only the relative change in fluorescence at 530 nm (excitation = 488 nm) is shown in Fig. 16 and resulted in a resting pH of 7.01 + 0.02 (n = 26, mean + SD. Cytoplasmic pH and Cazf in Human Platelets data is shown in Fig. 2. A shift in the entire population of platelets was observed upon addition of thrombin. A change in pH was observed at our first time point (15 s), the time to maximal change being dose-dependent; the time varied from 40 to 60 s. The broad population distribution in platelet resting internal pH is similar to that of resting [Ca2+]in; both are probably attributable to the broad platelet size distribution and to the variability in individual cell organellar contents. The thrombin dose dependence, as calculated by both quantitation techniques described above, is shown in Fig. 3; the curves are nearly superimposable and demonstrate a maximal pH change of 0.27 f 0.06 (n = 8, mean f SD., relative emission 530 nm) and 0.24 + 0.02 (n = 12, mean f S.D., 4881 454 excitation ratio) when a saturating dose of thrombin (9 Representative histogram of the ratio (cf. fig. 1) of BCECF-loaded platelets: resting (unshaded) and 40 s after stimulation with 0.05 units/ml (9 nM) thrombin (shaded, at maximal cytoplasmic alkalinization). The pH change was determined using the flow cytometer by calculating the difference between the maximal emission at 530 nm (excitation 488 nm) (0) and the ratios of excitation (488/454 nm) (Cl) before and after thrombin stimulation. The relative pH change was calculated from the appropriate calibration curve shown in Fig. 1, a and b. Maximal SE. for n 2 9, with three separate donors, was ~28%. For any one donor the S.E. was 8.3-11.1%. nM) was used as the stimulus. The large error bars reflect significant donor variability as well as the broad resting pHi, (Fig. 2). For any one donor, the standard error of the mean was much lower (8.3 to 11.1%) than that observed for four donors (n = 8, maximal SE. = 23%). Thus, flow cytometric analysis enables us to conclude that the thrombin-induced alkalinization is maximal within 60 s and that the time to maximal alkalinization and the final extent are both dosedependent.
We further investigated the rapid platelet acidification by amiloride pretreatment prior to thrombin stimulation. Since the acidification is extremely rapid and therefore is masked at saturating thrombin doses by the alkalinization, blockage of Na'/H' countertransport with amiloride permitted accurate quantitation of its dose dependence. Flow cytometry further permitted the determination of the presence or absence of subpopulations exhibiting acidification. Single cell analysis of the thrombin dose dependence of the acidification by flow cytometry and suspension analysis by fluorometry are shown in Fig. 4. Simultaneous experiments were performed on the cytometer and in suspension. The dose dependence was comparable by both techniques ( BCECF-loaded platelets' pH response to thrombin after pretreatment with 10m4 M dimethylamiloride was evaluated in suspension on the fluorometer and in single cells on the flow cytometer. The rate and the extent of the acidification were evaluated in suspension as slope/ 450 (0) and as the relative change in emission at 530 nm (0) (cf. "Methods"); the single cell FACS data were evaluated as emission 530 nm (excitation 488 nm only) (0) and as the excitation ratio (488/ 454 nm, emission 530 nm) (m). All data are presented as % of maximal response, 100% = maximal acidification (approximately 0.4 pH units).
Values are representative of one of three typical experiments each performed in duplicate. Platelets loaded with 1 PM BCECF were stimulated with thrombin (0.0025-0.05 units/ml, 0.45-9.0 nM) in the presence of dimethylamiloride (lo-* M, 2 min). The subsequent rate and extent of pH change were monitored. Data are presented as mean + S.D. for three separate exneriments (n 2 5).

Thrombin
Relative rate" acidification AF Platelets loaded simultaneously with 2 PM Indo-l and 1 pM BCECF were stimulated with 0.9 (0) and 9 nM (0) thrombin and their fluorescence emissions monitored continuously for 90 s. The Indo-l tracings (a) and the BCECF tracings (b) represent the time course after thrombin stimulation of the Ca2+ responding cells. Also included are tracings after pretreatment with 10e4 M amiloride for 2 min for 0.9 nM thrombin + amiloride (0) and 9.0 nM thrombin + amiloride (m). Data are representative tracings from one of three separate experiments. tion, maximal in less than 1 min, as the subsequent alkalinization is abolished. This pH change was observed in the entire platelet population, even at subsaturating doses of stimulus, and was dose-dependent with respect to both the rate and the extent of pH change. Simultaneous flow cytometric measurements of changes in cytoplasmic Ca2+ and H' concentrations permitted evaluation of the interdependence between the above pH changes and the Ca2+ transient in those subpopulations exhibiting the transient. Time course tracings of typical platelet responses to Lu-thrombin are shown in Fig. 5a ([Ca'+]J and Fig. 5b (pHi,). The appropriate controls using Indo-l and BCECF separately and then simultaneously indicated no overlapping of Indo-l and BCECF fluorescences. There was no appreciably detectable platelet autofluorescence. When 0.9 nM thrombin was used as the stimulus, the [Caz+]in rise was exhibited in only a subpopulation of the platelets (70%) as we have previously demonstrated (30), the Ca2+ rise being dose-inde-pendent, the proportion of cells responding being dose-dependent. In addition, platelets pretreated with amiloride prior to addition of thrombin (0.9, 9.0 nM) also exhibited a near normal, maximal Ca2+ rise, indicating that amiloride pretreatment did not affect the Ca2' fluxes. In all experiments, the maximal Ca*+ level was observed at the first time point, i.e. I5 s (due to instrumental limitations, earlier time points could not be attained). Fig. 5b demonstrates the relative pH change in these same, simultaneously analyzed thrombin-stimulated and amiloride pretreated, Ca2+ responding platelets. When 0.9 nM thrombin (a subsaturating dose) was used, a rapid acidification (0.10 pH units) was detected prior to the alkalinization. The alkalinization was dose-dependent (0.143 f 0.086 uersL(s 0.299 + 0.098 pH units for 0.9 and 9.0 nM cu-thrombin, respectively) and was maximal by 40-60 s. The acidification was not detectable in the 9.0 nM tracing, possibly due to masking of the much smaller acidification by the large rapid alkalinization. The thrombin-induced alkalinization was totally inhibited after preincubation with 10m4 M amiloride for 2 min (Fig.  5b) and the acidification was significantly prolonged. These data represent only the cells exhibiting a Ca2+ rise. We also analyzed the pH change in those cells not exhibiting a Ca*+ rise and found the pH curves to be identical. The acidification was therefore thrombin dose-dependent and exhibited in the entire population of platelets. Our results prevented us from drawing any definitive conclusions regarding the presence or absence of subpopulations exhibiting the alkalinization, since subsaturating thrombin concentrations induce an alkalinization too small to be analyzed by our present instrumentation (s-0.36 uers'sus ~0.15 pH units, for 0.9 nM + amiloride and 0.9 nM, respectively). Table II shows a summary of the simultaneous pH and Ca2+ data. The Ca2' transient was maximal in responding control and amiloride-treated platelets, and its magnitude was doseindependent. The percent of cells exhibiting a Ca2+ rise in control cells was dose-dependent, and, after amiloride pretreatment, there was a significant reduction in this percentage. This reduction is due to amiloride pretreatment and not due to a lower resting pH since addition of ammonium chloride (6 mM) to readjust the resting pH (after amiloride pretreatment) did not increase the percentage of the cells responding (data not shown). The alkalinization was thrombin dose-dependent in control platelets, as was the acidification in amiloride-treated platelets.

DISCUSSION
Flow cytometry permits responses by subpopulations of cells to be measured continuously. BCECF has been used previously to measure pH change on the FACS (33) as has Indo-l to measure Ca2+ (11,30,34), but this is the first report of simultaneous observations, permitting analysis of the interrelationships between [Ca'+]i,, transients and cytoplasmic pH changes. Flow cytometry offers several advantages over suspension fluorometric techniques including 1) a smaller number of cells required per run; 2) continuous monitoring of subpopulation responses without the intrinsic errors induced by probe leakage; and 3) simultaneous observation of several activation parameters. Limitations include the time required to stimulate, mix, and collect the first data point, usually lo- Platelets simultaneously loaded with Indo-l-AM (2 PM) and BCECF-AM (1 PM) were subsequently stimulated with varying doses of thrombin. Their relative intracellular pH and Ca*+ changes were monitored on the fluorescence activated cell sorter. The Ca*+ data presented are the maximal Ca2+ levels in the subpopulation exhibiting a change. The pH data are representative of the entire population. All values are represented as mean + S.D. (n 2 5). In many cases, especially at saturating thrombin doses when the rate of alkalinization was near maximal, the elapsed time prior to collection of the first time point was too long to permit observation of the initial rapid acidification. Since the acidification was obscured by the alkalinization, its dose dependence was evaluated after amiloride pretreatment.
The flow cytometric studies here are in accord with nonsimultaneous fluorometric suspension studies indicating a stimulus-induced alkalinization (from pH 7.0 to 7.3 with 24.5 nM thrombin) (4, 5, 7) and a rapid rise in [CaZ+]in (to 800 nM) (11,22). We here demonstrate the dose dependence of a rapid acidification, not easily visualized in suspension studies due to decreased fluorometric sensitivity and probe leakage problems, problems not encountered in single cell analyses. They also show, by simultaneous observations in the same platelet, that the Ca*+ transient was clearly maximal prior to the slower alkalinization.
After amiloride pretreatment, the acidification was prolonged and the Ca2+ rise was maximal, while the alkalinization and depolarization were totally abrogated, there was also a significant reduction in the percentage of cells exhibiting a Ca2+ rise. Since all cells observed exhibited a significant acidification, whether Ca2+ responding or not, the reduction in the percent of cells responding with a Ca*' rise could not be a result of cell death due to amiloride addition. These results also explain why other investigators, not able to examine Ca2+ subpopulations by flow cytometry and therefore analyzing the average of cells in suspension (responding and not responding), have reported a decreased magnitude of Ca*+ transient in the presence of amiloride. Our findings imply that the thrombin-induced acidification and Ca*+ transient are independent of the alkalinization and that the two H' fluxes observed upon thrombin stimulation are controlled differently.
Finally, previous studies (22) totally chelating intracellular Ca*+ demonstrated the presence of the acidification (and absence of subsequent alkalinization) after thrombin stimulation. We show here that this acidification was dose-dependent in a uniform platelet population (in contrast to the Ca*+ rise which was dose-independent and which occurred in a dose-dependent percentage of the population). It should be noted that cytoplasmic acidification is the only activation parameter, of those we have measured so far, that occurs in the absence of a membrane depolarization, a cytosolic alkalin-ization, and a rapid cytosolic [Ca2+]in rise. We conclude that not only is the alkalinization independent of the Ca2+ and H+ concentration increases but that the accumulation of cytoplasmic H' may be one of the early thrombin-induced activation signals.