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H+ exchange in human platelets activated with thrombin, arachidonic acid, A23187 and TPA
THROMBOSIS RESEARCH 55; 683-693, 1989 0049-3848/89 $3.00 + .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.
CYTOPLASMIC pH DEPENDENCY OF Na+/H+EXCHANGE IN HUMAN PLATELETS ACTIVATED WITH THROMBIN, ARACHIDONIC ACID, A23181 AND TPA
ATSUKO OGAWA, YOSHIHIDE ISHIKAWA and SHIGERU SASAKAWA The Japanese Red Cross Central Blood Center, 4-1-31, Hiroo, Shibuya-ku, Tokyo 150, Japan (Received
13.4.1989; accepted in revised form 12.6.1989 by Editor S. Okamoto)
ABSTRACT The cytoplasmic pH (pHc) of human platelets was lowered to 6.8-7.2 by treatment with various doses of nigericin (K/H+ ionophore), then these platelets were stimulated with thrombin, arachidonic acid (AA), A23187 or 12-0tetradecanoylphorbol-13-acetate (TPA), to monitor the pHc changes using a pH-sensitive fluorescent dye, BCECF. The pHc increased with the stimulation in an amiloridesensitive manner only when the resting-pHc was lower than a certain value (pHc 6.99 for thrombin-stimulation, 6.95 for AA, 7.04 for A23187 and 6.95 for TPA, n = 3). At a higher resting-pHc, the pHc decreased. Similar observations were found using platelets acid-loaded by Naacetate treatment. These results suggest that stimulation-induced activation of the Na+/H' exchanger depends on cytoplasmic H+ concentration, and it can function properly only when the resting-pHc is lower than about pHc 7.
INTRODUCTION Na+/H+exchanger is present in plasma membranes of many cell types. In unactivated human platelets, Na+/H+exchange is activated when the pHc is experimentally reduced by treatment with nigericin, a monovalent cation ionophore (11, or salts of weak acids including Na-propionate (2). It can be also activated by stimulating platelets with agonists, thrombin (1,3,4), a Ca'+ Since Na+/H'exionophore A23187 (51, and a phorbol ester (4).
Key words: platelet, activation, cytoplasmic pH, BCECF, Na+/H+ exchanger, pH dependency, 683
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change activity in dormant cells increases as the internal H+ concentration is raised, the Na+/H+ exchanger is considered to control pHc in resting cells (2,6). In stimulated cells, the Na+/H+ exchanger Is expected to play more active roles linked closely with stimulus-response processes. In epinephrine- and ADP-stimulated platelets, blockade of the Na+/H' exchanger inhibits phospholipase A1, subsequent phospholipase C-mediated reactions, secondary aggregation and secretion (7,8). Slffert and Akkerman have reported that activation of the Na+/H+ exchanger is an important signal in stimulus-response coupling and is an essential step in the cascade of events required to Increase cytoplasmic free Ca'+ in platelets (9). However, other investigators have shown that elevation of pHc or activation of Nat/H+exchange Is not an essential step in Ca*+mobilization in thrombin-stimulated platelets (10-13) and that it is not required for ADP-induced aggregation (14). Therefore the role of the Na'/H+exchanger in stimulated platelets remains to be clarified. Internal pH is an important parameter which determines the In cultured rat myoblast activity of the Na+/H+ exchanger. cells, the Na+/H'exchanger has pHc dependency and increases the sensitivity to cytoplasmic H' by stimulation with serum or TPA (15). In stimulated platelets, however, little is known about When internal H' the effect of pHc on the Na'/H+ exchanger. concentration affects the stimulation-induced Na+/H+ exchange, platelets having various resting-pHc are expected to show various rates of Na+/H+exchange and different patterns of pHc change by In this study, the effect of stimulation with an agonist. resting-pHc on agonist-induced changes of pHc was investigated by stimulating platelets with agonists after acid-loading.
MATERIALS AND METHODS Platelet preparation: Platelet concentrate (PC) was prepared from whole blood containing l/10 volume of CPD solution (16 mM citrate, 89 mM Na-citrate, 16 mM NaHIPOI and 129 mM dextrose) by a two-step centrifugation method (300 g for 10 min and 600 g for 15 mln) and stored overnight at 22°C with gentle agitation. Manipulation and determination of pHc: PC was incubated with 2'7'-bis(carboxyethyl)carboxyfluorescein tetraacetoxymethyl ester (BCECF-AM) (Wake Pure Chemicals, Japan) (6 PM) for 30 min at 22°C and centrifuged at 700-1000 g for 5 min with l/10 volume of citrate solution (75 mM Na-citrate, 38 mH citrate, pH 4.95). The precipitated platelets were resuspended in Hepes-buffered saline (135 mM NaCl, 5 mM KCl, 10 mM Hepes, pH 7.25 at 37°C). Agonists and reagents were dissolved in solvents as follows: bovine serum albumin (BSA) (Sigma Chemical Co.) and thrombin (Green Cross, Japan) in aqueous solution; arachidonic acid (AA) (Sigma), A23187 (Sigma), 12-o-tetradecanoylphorbol-13-acetate (TPA)(Sigma) and nigericin (Sigma) in ethanol; and amiloride (Sigma) in dimethylsulfoxlde. Platelets were acid-loaded by addition of nigericin or Na-acetate. Platelet suspension
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(3.5 x 10" platelets&l) was incubated at 37°C and mixed with reagents as follows: O-100 nM nigericin at 2 min, 5 mg/ml BSA at 2.5 min, and then 1 mM CaCll and an agonist at 4 min after the beginning of the incubation. Hepes buffer containing O-82 mM Na-acetate (10 mM Hepes, 5 mM KCl, NaCl and Na-acetate; total concentration of NaCl and Na-acetate was kept at 135 mM, pH 7.25 at 37 "C) was warmed to 37°C. After the platelet suspension (7.0 x 10' platelets/pi) had been preincubated at 37°C for 1 min and diluted with an equal volume of the warmed buffer, the platelets were stimulated by addition of 1 mM CaCll and an agonist 2 min after the dilution. In some experiments, amiloride (0.5 mM) was added 3 min before the stimulation. The final concentrations of ethanol and dimethylsulfoxide in the reaction mixture were less than 0.5% and 0.3%, respectively.
The fluorescence of the platelet suspension was monitored (hex = 500 nm, hem = 530 nm) at 37°C with a fluorospectrometer (JASCO, FP-550, Japan). Stirring of the platelet suspension was stopped just after the addition of an agonist. The measurements were repeated at a pH-insensitive excitation wavelength The fluorescence excited at (hex = 442 nm, hem = 530 nm). 500 nm was corrected by changes of the fluorescence excited at 442 nm and converted to the pHc value. The calibration of the fluorescence to pHc was carried out as described previously (16) except that platelets were suspended in 130 mt4 KC1 medium containing 10 mM NaCl and 10 mM Hepes (pH 7.25 at 37°C). Patterns of pHc changes (Fig. 1, 2 and 4) were obtained from measurement at hex = 500 nm and hem = 530 nm.
RESULTS Cytoplasmic acidification in resting platelets Platelets were treated with nigericin, an ionophore which transports K' and H' (Fig. 1-A). Platelet pHc decreased and this decrease could be slowed down by addition of concentrated BSA. Although BSA quenched the fluorescence of BCECF, the calculated pHc values were the same before and after the BSA addition. The pHc value was 7.18kO.04 (n = 16) just before addition of an agonist without nigericin treatment and decreased to 6.84fO.03 (n = 16) with 100 nM nigericin treatment. A salt of a weak acid, Na-acetate, was used as another tool for acid-loading of platelets (Fig. 1-B). Platelets suspended in the buffer without Na-acetate showed a gradual decrease of pHc to Platelet pHc 7.21f0.04 (n 7) just before agonist addition. rapidly acidified depending on the Na-acetate concentration, and then increased slowly when the acidified pHc was lower than 7.0750.02 (n = 3). The pHc rapidly fell to 6.8OkO.02 (n = 5) by treatment of platelets with 41 mM Na-acetate, and then gradually rose to 6.91f0.02 (n 7) just before addition of an agonist. The gradual alkalinization presumably reflected H'-extrusion by the Na+/H+ exchanger as reported by Livne et a1.(2) since amiloride blocked the alkalinization and caused; lower restingpHc (Fig. 4-B). ??
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Fiq. 1 Effect of nigericin and Na-acetate on pHc. CNa-metatel BCECP-loaded platelets were suspended in 10 mM Hepes buffer containing 135 mW NaCl and 5 mM KC1 (pH 7.25). (A) After preincubation of platelet suspension for 2 min at 37"C, O-100 nM nigericin imin (Nig) and 5 mg/ml BSA were mixed with the suspension, then, CaCL 1mM and 0.5 ~1 of ethanol (EtOH) were added. The scale for pHc is applicable only to the portion of the traces after BSA addition. (B) After preincubation for 1 min at 37 "C, platelet suspension was mixed with the Hepes buffer containing Na-acetate (O-41 mM) replacing NaCl (where indicated by arrow), then 1 mM of CaCL and 0.5 ~1 of ethanol (EtOH) were added to the suspension.
Changes of pHc induced by platelet activation Platelets treated with nigericin were stimulated by thrombin, AA, A23187 and TPA, and their aggregability was measured in the presence of BSA. Since platelets decreased in sensitivity to AA and A23187 in the presence of BSA to about l/20 and l/4, respectively, agonist doses used for stimulation were determined as follows: thrombin, AA and A23187 were used at concentrations ranging from the minimum dose that induced maximum aggregation to TPA was used in a dose that a dose double this minimum dose. induced 30-50% aggregation at 3 min. Platelets treated with 100 104 nigericin has been found to show increased sensitivity to thrombin, A23187 and TPA, but decreased sensitivity to AA (16). In present condition, treatment of 100 nM nigericin increased aggregation from 37% to 53% (at 3 min, n = 3) in TPA-stimulation and decreased it from 60% (maximum aggregation) to 27% (n = 3) The aggregability could not increase in in M-stimulation. thrombin- and A23187-stimulation since it was maximum (about 80%) without nigericin treatment. Platelet stimulation induced pHc changes (Fig. 2-A). Platelets showed the largest changes by stimulation with A23187 and the smallest changes by stimulation with TPA. With all four agonists, pHc decreased when the resting-pHc was higher, but it increased to a maximum at 20-60 set after the addition of the agonist when the resting-pHc was lower. Although 0.5 mM of amiloride hardly affected the decrease of pHc in platelets having a resting-pHc of 7.1-7.2 (data not shown), it blocked the increase of pHc (Pig. 2-B). Platelets suspended in a Na-free buffer (that contained choline-Cl or N-methylglucamine-Cl instead of NaCl) did not show any agonist-induced increase of pHc (data not shown). The value of pHc after stimulation was plotted against each resting-pHc just before the stimulation (Fig. 3). Since pHc changed slightly even without stimulation as shown in Fig. l-A, pHc after addition of ethanol was also plotted against
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2 in
Changes of nigericintreated platelets stimulation. Only traces for the
platelets were stimul lated with 33 mu/ml thrombin (Thr), 1 1 100 FM AA, 333 nM 1 6.86-~--6.83-\___~ 6.9o-L_4_ A23181 and 25 nM TPA in the presence of 1 mM CaCl,. The figure at each trace indicates the calculated PHC value just before the stimulation. (B) Amiloride (0.5 mM) was added to the platelet suspension 3 min before the stimulation.
symbol (O,A,o) represents an individual experiment. Platelet pHc was also determined after addition of 0.5 ~1 ethanol instead of an agonist (*). Resting
pHc
the resting pHc, and straight line was obtained. In the case of platelets activated by an agonist, for example, with thrombin (Fig. 3-A), the pHc decreased when the resting-pHc was higher than 6.99, but increased at a lower resting-pHc. After stimulation, pHc reached a constant value (pHc 6.99) with a definite range of resting-pHc (pHc 6.9-7.1). There was a resting-pHc at which changes of pHc by stimulation was minimum. The resting-pHc is termed the *'apparent set point", because pHc appeared to be The apparent set point was determined as the set for the PH. intersection of two lines obtained from changes of pHc with and
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Changes of Fig. 4. pHc in Na-acetatetreated platelets after stimulation. drawn Traces were the part only for 0.5 min before addition of an agonist.
thrombin 5 CIM AA, B I 1 1 1 83 nM A23187 and 6.0!36.81-Y+-+6.66~ 6804-25 nM TPA in the The figure at each trace indicates the presence of 1 mM CaCL. calculated pHc value just before the stimulation. (B) Platelets were treated with amiloride (0.5 mM) 3 min before stimulation and treated with 41 mM Na-acetate. 25
mu/ml
(Thr),
Fig. 5. Effect of restingPHC in Na-acetate-treated platelets on pHc changes induced by stimulation. After treatment with Naacetate, platelets were stimulated with 25 mu/ml thrombin (A), 5 FM AA (B), 83 nM A23187 (C) and 25 nM TPA (D) in the presence of 1 mM CaCL. The pHc values were after stimulation determined at 30 set (AA, A23187), 45 set (thrombin) or 90 set (TPA) after the Resting pi-k addition of the agonists (indicated by arrowheads in represents an individual Fig. 4-A). Each type of symbol (o,a,o) Platelet pHc was also measured after addition of experiment. 0.5 ul ethanol instead of an agonist (0).
without stimulation. Platelet pHc also changed similarly by stimulation with AA, A23187 and TPA. The apparent set points were 6.99f0.03 with 33 mu/ml of thrombin-stimulation, 6.95t0.01 with 100 PM AA, 7.04kO.01 with 333 nM A23187 and 6.95kO.02 with 25 nM TPA (n = 3). When platelets were stimulated by half of the amount of the agonists described above, the apparent set point did not differ although each pHc change decreased (data not shown). The apparent set point was also determined to be pHc 7.08 (n = 2) and 7.03 (n = 2) by stimulating platelets with 10 mu/ml thrombin and 63 uM AA, respectively, using platelets prepared from fresh platelet-rich-plasma within 6 hr of collec-
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tion. To confirm that the effects of nigericin on agonist-induced changes of pHc were results of diverse resting-pHc values, similar experiments were performed using platelets acid-loaded with Na-acetate (Fig. 4, 5). Although their pHc values changed gradually after acid-loading as shown in Fig. l-B, the pHc also changed by stimulation. The stimulation-induced changes of pHc were similar to those using the platelets treated with nigericin, i.e., A23187 caused the largest changes and TPA caused the smallest changes (Pig. 4-A). The pHc decreased from the higher resting-pHc but increased from the lower resting-pHc. Amiloride inhibited the elevation of pHc induced by both the acid-loading with Na-acetate and the stimulation with agonist (Fig. 4-B). Apparent set points were 7.06f0.01 with 25 mu/ml thrombinstimulation, 6.99kO.03 with 5 PM AA, 7.08f0.01 with 83 nM A23187 and 7.07iO.02 with 25 nM TPA (n = 3) (Fig. 5). After the same platelet preparation had been treated with nigericin or Na-acetate, apparent set points were determined to compare the two treatments. Apparent set points of the platelets treated with nigericin and Na-acetate were; 6.95 and 7.01 with 25 mu/ml thrombin; 7.01 with 50 pM AA and 7.00 with 5 uM AA; 7.06 with 333 nM A23187 and 7.07 with 83 nM A23187; and 6.89 and 7.03 with 25 nM TPA, respectively. Consequently, apparent set points were the same by the two treatments in AA- and A23187-stimulated platelets. In thrombin- and TPA-stmulated platelets, however, the values were higher with the Na-acetate treatment than the nigericin treatment.
DISCUSSION After platelet stimulation, pHc increased from a low resting-pHc, but decreased from a high resting-pHc in the platelets which had been treated with nigericin or Na-acetate. This phenomenon was observed both in platelets stored overnight, which were mainly used, and in fresh platelets within 6 hr of collection. Amiloride, an inhibitor of Na+/H+ exchanger, blocked pHc increase, but not its decrease, which had been induced by stimulation. This result suggests that stimulation-induced increase of pHc was caused by activation of the Na+/H+ exchanger at the low resting-pHc, and that the Na+/H+ exchanger Is inactive at In resting cells, the activhigh resting-pHc (above pHc 7.1). ity of Na+/H+ exchange is not detectable at high pHc and increases with lowering of the pHc in a slgmoidal manner (2,6,15), and the pHc-sensitivity results from the allosteric activation of Therefore, the Na+/H+ exchanger by the substrate H+ (17). agonist-induced Na+/H+ exchange is sensitive to pHc, like the Na+/H+ exchange in resting platelets (21, and can be activated sufficiently only when the pHc is lower than a certain value. We have reported in a previous paper that the resting-pHc is about 7.15 in platelets treated with BCECF-AM in plasma and about 6.95 in those treated with BCECF-AM in buffer (16). This differ-
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ence in the pHc resulted partly from its gradual decrease when the platelets were suspended in buffer (unpublished result 1. We loaded the platelets with BCECF in plasma to prevent cell damage, but other researchers have loaded them with BCECP in buffer (1,2, probably had higher pHc than 4,9,10,12,13). Thus, our platelets those used in these other studies, and this may be one reason for our obsevation of a pHc decrease while others have reported stimulation-induced increases (1,3-5,9,10,12,13). The decrease of pHc is observed by stimulating platelets whose Na+/H+ exchange has been blocked (1,12-141, which can result from an increase in H+ production by stimulation, especially by the Caa+-related reaction (e.g., increased myosin ATPase activity) (1). Consequently, pHc changes can be explained as follows: In platelets having high resting-pHc, stimulation-induced decrease of pHc is caused by accumulating H+ in the cytoplasm since Na+/H+ exchange is inactive at high pHc. The activity of the Na+/H+ exchanger increases with decreasing pHc, resulting in compensation for the stimulation-induced decrease of pHc. When the resting-pHc was lower than the definite value, Nat/H+ exchanger was fully activated, and this caused the pHc to rise after stimulation. In the platelets having resting-pHc of 6.9-7.1, the pHc changed to a constant value by stimulation. The apparent set point was determined as an indication of the constant value. Among the four agonists used here, the apparent set point was the lowest with M-stimulation, while it was the highest with A23187stimulation. The apparent set point probably arises from a balance of the decrease of pHc by H+ production and the increase of pHc by H’ extrusion via Na+/H+ exchanger. Since the apparent set point did not differ with agonist dose, the differences in the apparent set point were not due to differences in the lntensity of stimulation. They may be caused by differences in the balance of the reaction systems activated by each agonist. Caa+dependent reaction seems to contribute to activation of H’ production (11, while C-kinase may be responsible for activation of Na+/H+ exchange (15,181. As there has been no other detailed study on these activation mechanisms, further investigation is required to find whether the diversity of apparent set point among the agonists depends on the activation of the Na+/H+ exchanger or on the activation of the H’ production. Vigne -et a1.(15) have reported that Na+/H+ exchanger in rat myoblast cells sifts the pHc dependence toward alkaline pHc values and increases the maximal activity at acidic pHc by cell stimulation, using “Na+ uptake techniques for measurement of the exchange activity. In resting platelets, Na+/H+ exchanger was activated when pHc was acidified below 7.07 by Na-acetate treatment (Fig. 1-B). The value was close to the apparent set points determined by stimulating Na-acetate-treated platelets with thromin, A23187 and TPA (Fig. 5). Thus, a difference In pHc dependence of the exchangers In resting platelets and in stimulated platelets can not be detected by the measurement of the pHc changes. Differences in the characteristics of the exchangers In resting and stimulated platelets may be clarified by using a method, such as rzNa+ uptake technique, determining the exchange activity directly.
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Since the Nat/H+ exchange is not activated in platelets having high resting-pHc, its activation does not seem to be an indispensable step for Caa+ release in the stimulus-response processes of platelets as described by Siffert and Akkerman (9). Elevation of pHc, however, increases Ca'*-release (9,161 and phospholipase Al activity (7) induced by stimulation, especially with a low dose of agonists. A lowering of the pHc inhibits the metabolism of AA to thromboxan AI in AA-stimulated platelets Phospholipase A1 has pH dependency and increases the (19). sensitivity to Ca'+ with elevation of the pH in vitro (20). Therefore, in platelets having low resting-pHc, elevation of pHc by the Na+/H+ exchanger probably plays a more important role. These findings also suggested that pHc modulates the activity of intracellular reactions, especially when platelets are stimulated with thresholds of agonist doses. Consequently, the stimulationactivated Na+/H+ exchanger is considered to elevate the pHc or prevent its stimulation-induced decrease to maintain the pHc at a constant value and thus modulate intracellular reactions during platelet stimulation.
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EAVOICO, G.B., CRAGOE, E.J.,Jr. and PEINSTEIN, M.B. Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+exchange and its inhibition by amiloride analogs. 2. -Biol. Chem. 261, 13160-13167, 1986.-
2.
LIVNE, A., GRINSTEIN, S. and ROTHSTEIN, A. Characterization of Na+/H' exchange in platelets. Thromb. Haemostas. 58, 971-977, 1987.
3.
SIPFERT, W., FOX. G., MUCKENHOFF, K. and SCHEID, P. Thrombin stimulates Na+-H+ exchange across the human platelet plasma membrane. --FEBS Lett. 172, 272-274, 1984.
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SIEFFERT, W., SIFFERT, G. and SCHEID. P. Activation of Na+/H+ exchange in human platelets stimulated by thrombin and a phorbol ester. Biochem. J. 241, 301-303, 1987.
5.
SIFFERT, W., MUCKENHOFF, K. and SCHEID, P. Evidence for a role of Na*/H+exchange in platelets activated with calciumionophore A23187. Biochem. Biophys. Res. Commun. 125, 1123-1128, 1984.
6.
GRINSTEIN, S., COHEN, S. and ROTHSTEIN, A. Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H'antiport. 2. Gen. Physiol. 83, 341-369, 1984.
7.
SWEATT, J.D., CONNOLLY, T.M., CRAGOE, E.J. and LIMBIRD, L.E. Evidence that Na'/H+ exchange regulates receptor-mediated phospholipase AZ activation in human platelets. 2. Biol. Chem. 261, 8667-8673, 1986.
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8.
BANGA, H.S., SIMONS, E.R., BRASS, L.F. and RITTENHOUSE, S.E. Activation of phospholipases A and C in human platelets exposed to epinephrine: Role of glycoproteins IIb/IIIa and Proc. Natl. Acad. Sci. USA. 83, dual role of epinephrine. ----9197-9201, 1986.
9.
SIFPERT, W. and AKKERMAN, J.W.N. Activation of sodiumproton exchange is a prerequisite for Ca'+ mobilization in human platelets. Nature, 325, 456-458, 1987.
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SIMPSON, A.W.M. and RINK, T.J. Elevation of pHi is not an essential step in calcium mobilisation in fura-2-loaded human platelets. -FEBS Lett. 222, 144-148, 1987.
11.
HUNYADY, L., SARKADI, B., CRAGOE, E.J.Jr. SPAT, A. and GARDOS, G. Activation of sodium-proton exchange is not a prerequisite for Ca'+mobilization and aggregation in human platelets. -FEBS Lett. 225, 72-76, 1987.
12.
SANCHEZ, A., ALONSO, M.T. and COLLAZOS, J.M. Thrombininduced changes of intracellular [Ca'+l and pH in human platelets. Cytoplasmic alkalinization is not a prerequisite for calcium mobilization. Biochim. Biophys. Acta 938, 497-500, 1988.
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ZAVOICO, G.B. and CRAGOE, E.J.,Jr. Ca" mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets. 2. -Biol. Chem-. 263, 9635-9639, 1988.
14.
FUNDER, J., HERSHCO, L., ROTHSTEIN, A. and LIVNE, A. Na+/H+ exchange and aggregation of human platelets activated by ADP: the exchange is not required for aggregation. Biochim. Biophys. Acta 938, 425-433, 1988.
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VIGNE, P., FRELIN, C. and LAZDUNSKI, M. The Na+/H+antiport is activated by serum and phorbol esters in proliferating myoblasts but not in differentiated myotubes. Properties of the activation process. 2. --Biol. Chem. 260, 8008-8013, 1985.
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OGAWA, A., ISHIKAWA, Y. and SASAKAWA, S. Effects of changes in intracellular pH induced by platelet storage and treatment with monovalent cation ionophores on platelet functions. Thromb. Res. 52, 369-379, 1988.
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ARONSON, P.S., NEE, J. and SUHM, M.A. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature, 299, 161-163, 1982.
18.
SIFFERT, W. and AKKERMAN, J.W.N. Protein kinase C enhances Ca'+ mobilization in human platelets by activating Na+/H+ exchange. 2. Biol. Chem. 263, 4223-4227, 1988.
19.
ISHIKAWA, Y., OGAWA, A. and SASAKAWA, S. Effect of cytoplasmic pH on platelet activation by arachidonic acid.
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Thromb. Res. 1989, in press. 20.
BARON, B.M. and LIMBIRD, L.E. Human platelet phospholipase AI activity is responsive in vitro to pH and Cal+variations which parallel those occurring after platelet activation in vivo. Biochim. Biophys. Acta, 971, 103-111, 1988.
CYTOPLASMIC pH DEPENDENCY OF Na+/H+EXCHANGE IN HUMAN PLATELETS ACTIVATED WITH THROMBIN, ARACHIDONIC ACID, A23181 AND TPA
ATSUKO OGAWA, YOSHIHIDE ISHIKAWA and SHIGERU SASAKAWA The Japanese Red Cross Central Blood Center, 4-1-31, Hiroo, Shibuya-ku, Tokyo 150, Japan (Received
13.4.1989; accepted in revised form 12.6.1989 by Editor S. Okamoto)
ABSTRACT The cytoplasmic pH (pHc) of human platelets was lowered to 6.8-7.2 by treatment with various doses of nigericin (K/H+ ionophore), then these platelets were stimulated with thrombin, arachidonic acid (AA), A23187 or 12-0tetradecanoylphorbol-13-acetate (TPA), to monitor the pHc changes using a pH-sensitive fluorescent dye, BCECF. The pHc increased with the stimulation in an amiloridesensitive manner only when the resting-pHc was lower than a certain value (pHc 6.99 for thrombin-stimulation, 6.95 for AA, 7.04 for A23187 and 6.95 for TPA, n = 3). At a higher resting-pHc, the pHc decreased. Similar observations were found using platelets acid-loaded by Naacetate treatment. These results suggest that stimulation-induced activation of the Na+/H' exchanger depends on cytoplasmic H+ concentration, and it can function properly only when the resting-pHc is lower than about pHc 7.
INTRODUCTION Na+/H+exchanger is present in plasma membranes of many cell types. In unactivated human platelets, Na+/H+exchange is activated when the pHc is experimentally reduced by treatment with nigericin, a monovalent cation ionophore (11, or salts of weak acids including Na-propionate (2). It can be also activated by stimulating platelets with agonists, thrombin (1,3,4), a Ca'+ Since Na+/H'exionophore A23187 (51, and a phorbol ester (4).
Key words: platelet, activation, cytoplasmic pH, BCECF, Na+/H+ exchanger, pH dependency, 683
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change activity in dormant cells increases as the internal H+ concentration is raised, the Na+/H+ exchanger is considered to control pHc in resting cells (2,6). In stimulated cells, the Na+/H+ exchanger Is expected to play more active roles linked closely with stimulus-response processes. In epinephrine- and ADP-stimulated platelets, blockade of the Na+/H' exchanger inhibits phospholipase A1, subsequent phospholipase C-mediated reactions, secondary aggregation and secretion (7,8). Slffert and Akkerman have reported that activation of the Na+/H+ exchanger is an important signal in stimulus-response coupling and is an essential step in the cascade of events required to Increase cytoplasmic free Ca'+ in platelets (9). However, other investigators have shown that elevation of pHc or activation of Nat/H+exchange Is not an essential step in Ca*+mobilization in thrombin-stimulated platelets (10-13) and that it is not required for ADP-induced aggregation (14). Therefore the role of the Na'/H+exchanger in stimulated platelets remains to be clarified. Internal pH is an important parameter which determines the In cultured rat myoblast activity of the Na+/H+ exchanger. cells, the Na+/H'exchanger has pHc dependency and increases the sensitivity to cytoplasmic H' by stimulation with serum or TPA (15). In stimulated platelets, however, little is known about When internal H' the effect of pHc on the Na'/H+ exchanger. concentration affects the stimulation-induced Na+/H+ exchange, platelets having various resting-pHc are expected to show various rates of Na+/H+exchange and different patterns of pHc change by In this study, the effect of stimulation with an agonist. resting-pHc on agonist-induced changes of pHc was investigated by stimulating platelets with agonists after acid-loading.
MATERIALS AND METHODS Platelet preparation: Platelet concentrate (PC) was prepared from whole blood containing l/10 volume of CPD solution (16 mM citrate, 89 mM Na-citrate, 16 mM NaHIPOI and 129 mM dextrose) by a two-step centrifugation method (300 g for 10 min and 600 g for 15 mln) and stored overnight at 22°C with gentle agitation. Manipulation and determination of pHc: PC was incubated with 2'7'-bis(carboxyethyl)carboxyfluorescein tetraacetoxymethyl ester (BCECF-AM) (Wake Pure Chemicals, Japan) (6 PM) for 30 min at 22°C and centrifuged at 700-1000 g for 5 min with l/10 volume of citrate solution (75 mM Na-citrate, 38 mH citrate, pH 4.95). The precipitated platelets were resuspended in Hepes-buffered saline (135 mM NaCl, 5 mM KCl, 10 mM Hepes, pH 7.25 at 37°C). Agonists and reagents were dissolved in solvents as follows: bovine serum albumin (BSA) (Sigma Chemical Co.) and thrombin (Green Cross, Japan) in aqueous solution; arachidonic acid (AA) (Sigma), A23187 (Sigma), 12-o-tetradecanoylphorbol-13-acetate (TPA)(Sigma) and nigericin (Sigma) in ethanol; and amiloride (Sigma) in dimethylsulfoxlde. Platelets were acid-loaded by addition of nigericin or Na-acetate. Platelet suspension
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(3.5 x 10" platelets&l) was incubated at 37°C and mixed with reagents as follows: O-100 nM nigericin at 2 min, 5 mg/ml BSA at 2.5 min, and then 1 mM CaCll and an agonist at 4 min after the beginning of the incubation. Hepes buffer containing O-82 mM Na-acetate (10 mM Hepes, 5 mM KCl, NaCl and Na-acetate; total concentration of NaCl and Na-acetate was kept at 135 mM, pH 7.25 at 37 "C) was warmed to 37°C. After the platelet suspension (7.0 x 10' platelets/pi) had been preincubated at 37°C for 1 min and diluted with an equal volume of the warmed buffer, the platelets were stimulated by addition of 1 mM CaCll and an agonist 2 min after the dilution. In some experiments, amiloride (0.5 mM) was added 3 min before the stimulation. The final concentrations of ethanol and dimethylsulfoxide in the reaction mixture were less than 0.5% and 0.3%, respectively.
The fluorescence of the platelet suspension was monitored (hex = 500 nm, hem = 530 nm) at 37°C with a fluorospectrometer (JASCO, FP-550, Japan). Stirring of the platelet suspension was stopped just after the addition of an agonist. The measurements were repeated at a pH-insensitive excitation wavelength The fluorescence excited at (hex = 442 nm, hem = 530 nm). 500 nm was corrected by changes of the fluorescence excited at 442 nm and converted to the pHc value. The calibration of the fluorescence to pHc was carried out as described previously (16) except that platelets were suspended in 130 mt4 KC1 medium containing 10 mM NaCl and 10 mM Hepes (pH 7.25 at 37°C). Patterns of pHc changes (Fig. 1, 2 and 4) were obtained from measurement at hex = 500 nm and hem = 530 nm.
RESULTS Cytoplasmic acidification in resting platelets Platelets were treated with nigericin, an ionophore which transports K' and H' (Fig. 1-A). Platelet pHc decreased and this decrease could be slowed down by addition of concentrated BSA. Although BSA quenched the fluorescence of BCECF, the calculated pHc values were the same before and after the BSA addition. The pHc value was 7.18kO.04 (n = 16) just before addition of an agonist without nigericin treatment and decreased to 6.84fO.03 (n = 16) with 100 nM nigericin treatment. A salt of a weak acid, Na-acetate, was used as another tool for acid-loading of platelets (Fig. 1-B). Platelets suspended in the buffer without Na-acetate showed a gradual decrease of pHc to Platelet pHc 7.21f0.04 (n 7) just before agonist addition. rapidly acidified depending on the Na-acetate concentration, and then increased slowly when the acidified pHc was lower than 7.0750.02 (n = 3). The pHc rapidly fell to 6.8OkO.02 (n = 5) by treatment of platelets with 41 mM Na-acetate, and then gradually rose to 6.91f0.02 (n 7) just before addition of an agonist. The gradual alkalinization presumably reflected H'-extrusion by the Na+/H+ exchanger as reported by Livne et a1.(2) since amiloride blocked the alkalinization and caused; lower restingpHc (Fig. 4-B). ??
??
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Fiq. 1 Effect of nigericin and Na-acetate on pHc. CNa-metatel BCECP-loaded platelets were suspended in 10 mM Hepes buffer containing 135 mW NaCl and 5 mM KC1 (pH 7.25). (A) After preincubation of platelet suspension for 2 min at 37"C, O-100 nM nigericin imin (Nig) and 5 mg/ml BSA were mixed with the suspension, then, CaCL 1mM and 0.5 ~1 of ethanol (EtOH) were added. The scale for pHc is applicable only to the portion of the traces after BSA addition. (B) After preincubation for 1 min at 37 "C, platelet suspension was mixed with the Hepes buffer containing Na-acetate (O-41 mM) replacing NaCl (where indicated by arrow), then 1 mM of CaCL and 0.5 ~1 of ethanol (EtOH) were added to the suspension.
Changes of pHc induced by platelet activation Platelets treated with nigericin were stimulated by thrombin, AA, A23187 and TPA, and their aggregability was measured in the presence of BSA. Since platelets decreased in sensitivity to AA and A23187 in the presence of BSA to about l/20 and l/4, respectively, agonist doses used for stimulation were determined as follows: thrombin, AA and A23187 were used at concentrations ranging from the minimum dose that induced maximum aggregation to TPA was used in a dose that a dose double this minimum dose. induced 30-50% aggregation at 3 min. Platelets treated with 100 104 nigericin has been found to show increased sensitivity to thrombin, A23187 and TPA, but decreased sensitivity to AA (16). In present condition, treatment of 100 nM nigericin increased aggregation from 37% to 53% (at 3 min, n = 3) in TPA-stimulation and decreased it from 60% (maximum aggregation) to 27% (n = 3) The aggregability could not increase in in M-stimulation. thrombin- and A23187-stimulation since it was maximum (about 80%) without nigericin treatment. Platelet stimulation induced pHc changes (Fig. 2-A). Platelets showed the largest changes by stimulation with A23187 and the smallest changes by stimulation with TPA. With all four agonists, pHc decreased when the resting-pHc was higher, but it increased to a maximum at 20-60 set after the addition of the agonist when the resting-pHc was lower. Although 0.5 mM of amiloride hardly affected the decrease of pHc in platelets having a resting-pHc of 7.1-7.2 (data not shown), it blocked the increase of pHc (Pig. 2-B). Platelets suspended in a Na-free buffer (that contained choline-Cl or N-methylglucamine-Cl instead of NaCl) did not show any agonist-induced increase of pHc (data not shown). The value of pHc after stimulation was plotted against each resting-pHc just before the stimulation (Fig. 3). Since pHc changed slightly even without stimulation as shown in Fig. l-A, pHc after addition of ethanol was also plotted against
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687
2 in
Changes of nigericintreated platelets stimulation. Only traces for the
platelets were stimul lated with 33 mu/ml thrombin (Thr), 1 1 100 FM AA, 333 nM 1 6.86-~--6.83-\___~ 6.9o-L_4_ A23181 and 25 nM TPA in the presence of 1 mM CaCl,. The figure at each trace indicates the calculated PHC value just before the stimulation. (B) Amiloride (0.5 mM) was added to the platelet suspension 3 min before the stimulation.
symbol (O,A,o) represents an individual experiment. Platelet pHc was also determined after addition of 0.5 ~1 ethanol instead of an agonist (*). Resting
pHc
the resting pHc, and straight line was obtained. In the case of platelets activated by an agonist, for example, with thrombin (Fig. 3-A), the pHc decreased when the resting-pHc was higher than 6.99, but increased at a lower resting-pHc. After stimulation, pHc reached a constant value (pHc 6.99) with a definite range of resting-pHc (pHc 6.9-7.1). There was a resting-pHc at which changes of pHc by stimulation was minimum. The resting-pHc is termed the *'apparent set point", because pHc appeared to be The apparent set point was determined as the set for the PH. intersection of two lines obtained from changes of pHc with and
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Changes of Fig. 4. pHc in Na-acetatetreated platelets after stimulation. drawn Traces were the part only for 0.5 min before addition of an agonist.
thrombin 5 CIM AA, B I 1 1 1 83 nM A23187 and 6.0!36.81-Y+-+6.66~ 6804-25 nM TPA in the The figure at each trace indicates the presence of 1 mM CaCL. calculated pHc value just before the stimulation. (B) Platelets were treated with amiloride (0.5 mM) 3 min before stimulation and treated with 41 mM Na-acetate. 25
mu/ml
(Thr),
Fig. 5. Effect of restingPHC in Na-acetate-treated platelets on pHc changes induced by stimulation. After treatment with Naacetate, platelets were stimulated with 25 mu/ml thrombin (A), 5 FM AA (B), 83 nM A23187 (C) and 25 nM TPA (D) in the presence of 1 mM CaCL. The pHc values were after stimulation determined at 30 set (AA, A23187), 45 set (thrombin) or 90 set (TPA) after the Resting pi-k addition of the agonists (indicated by arrowheads in represents an individual Fig. 4-A). Each type of symbol (o,a,o) Platelet pHc was also measured after addition of experiment. 0.5 ul ethanol instead of an agonist (0).
without stimulation. Platelet pHc also changed similarly by stimulation with AA, A23187 and TPA. The apparent set points were 6.99f0.03 with 33 mu/ml of thrombin-stimulation, 6.95t0.01 with 100 PM AA, 7.04kO.01 with 333 nM A23187 and 6.95kO.02 with 25 nM TPA (n = 3). When platelets were stimulated by half of the amount of the agonists described above, the apparent set point did not differ although each pHc change decreased (data not shown). The apparent set point was also determined to be pHc 7.08 (n = 2) and 7.03 (n = 2) by stimulating platelets with 10 mu/ml thrombin and 63 uM AA, respectively, using platelets prepared from fresh platelet-rich-plasma within 6 hr of collec-
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tion. To confirm that the effects of nigericin on agonist-induced changes of pHc were results of diverse resting-pHc values, similar experiments were performed using platelets acid-loaded with Na-acetate (Fig. 4, 5). Although their pHc values changed gradually after acid-loading as shown in Fig. l-B, the pHc also changed by stimulation. The stimulation-induced changes of pHc were similar to those using the platelets treated with nigericin, i.e., A23187 caused the largest changes and TPA caused the smallest changes (Pig. 4-A). The pHc decreased from the higher resting-pHc but increased from the lower resting-pHc. Amiloride inhibited the elevation of pHc induced by both the acid-loading with Na-acetate and the stimulation with agonist (Fig. 4-B). Apparent set points were 7.06f0.01 with 25 mu/ml thrombinstimulation, 6.99kO.03 with 5 PM AA, 7.08f0.01 with 83 nM A23187 and 7.07iO.02 with 25 nM TPA (n = 3) (Fig. 5). After the same platelet preparation had been treated with nigericin or Na-acetate, apparent set points were determined to compare the two treatments. Apparent set points of the platelets treated with nigericin and Na-acetate were; 6.95 and 7.01 with 25 mu/ml thrombin; 7.01 with 50 pM AA and 7.00 with 5 uM AA; 7.06 with 333 nM A23187 and 7.07 with 83 nM A23187; and 6.89 and 7.03 with 25 nM TPA, respectively. Consequently, apparent set points were the same by the two treatments in AA- and A23187-stimulated platelets. In thrombin- and TPA-stmulated platelets, however, the values were higher with the Na-acetate treatment than the nigericin treatment.
DISCUSSION After platelet stimulation, pHc increased from a low resting-pHc, but decreased from a high resting-pHc in the platelets which had been treated with nigericin or Na-acetate. This phenomenon was observed both in platelets stored overnight, which were mainly used, and in fresh platelets within 6 hr of collection. Amiloride, an inhibitor of Na+/H+ exchanger, blocked pHc increase, but not its decrease, which had been induced by stimulation. This result suggests that stimulation-induced increase of pHc was caused by activation of the Na+/H+ exchanger at the low resting-pHc, and that the Na+/H+ exchanger Is inactive at In resting cells, the activhigh resting-pHc (above pHc 7.1). ity of Na+/H+ exchange is not detectable at high pHc and increases with lowering of the pHc in a slgmoidal manner (2,6,15), and the pHc-sensitivity results from the allosteric activation of Therefore, the Na+/H+ exchanger by the substrate H+ (17). agonist-induced Na+/H+ exchange is sensitive to pHc, like the Na+/H+ exchange in resting platelets (21, and can be activated sufficiently only when the pHc is lower than a certain value. We have reported in a previous paper that the resting-pHc is about 7.15 in platelets treated with BCECF-AM in plasma and about 6.95 in those treated with BCECF-AM in buffer (16). This differ-
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ence in the pHc resulted partly from its gradual decrease when the platelets were suspended in buffer (unpublished result 1. We loaded the platelets with BCECF in plasma to prevent cell damage, but other researchers have loaded them with BCECP in buffer (1,2, probably had higher pHc than 4,9,10,12,13). Thus, our platelets those used in these other studies, and this may be one reason for our obsevation of a pHc decrease while others have reported stimulation-induced increases (1,3-5,9,10,12,13). The decrease of pHc is observed by stimulating platelets whose Na+/H+ exchange has been blocked (1,12-141, which can result from an increase in H+ production by stimulation, especially by the Caa+-related reaction (e.g., increased myosin ATPase activity) (1). Consequently, pHc changes can be explained as follows: In platelets having high resting-pHc, stimulation-induced decrease of pHc is caused by accumulating H+ in the cytoplasm since Na+/H+ exchange is inactive at high pHc. The activity of the Na+/H+ exchanger increases with decreasing pHc, resulting in compensation for the stimulation-induced decrease of pHc. When the resting-pHc was lower than the definite value, Nat/H+ exchanger was fully activated, and this caused the pHc to rise after stimulation. In the platelets having resting-pHc of 6.9-7.1, the pHc changed to a constant value by stimulation. The apparent set point was determined as an indication of the constant value. Among the four agonists used here, the apparent set point was the lowest with M-stimulation, while it was the highest with A23187stimulation. The apparent set point probably arises from a balance of the decrease of pHc by H+ production and the increase of pHc by H’ extrusion via Na+/H+ exchanger. Since the apparent set point did not differ with agonist dose, the differences in the apparent set point were not due to differences in the lntensity of stimulation. They may be caused by differences in the balance of the reaction systems activated by each agonist. Caa+dependent reaction seems to contribute to activation of H’ production (11, while C-kinase may be responsible for activation of Na+/H+ exchange (15,181. As there has been no other detailed study on these activation mechanisms, further investigation is required to find whether the diversity of apparent set point among the agonists depends on the activation of the Na+/H+ exchanger or on the activation of the H’ production. Vigne -et a1.(15) have reported that Na+/H+ exchanger in rat myoblast cells sifts the pHc dependence toward alkaline pHc values and increases the maximal activity at acidic pHc by cell stimulation, using “Na+ uptake techniques for measurement of the exchange activity. In resting platelets, Na+/H+ exchanger was activated when pHc was acidified below 7.07 by Na-acetate treatment (Fig. 1-B). The value was close to the apparent set points determined by stimulating Na-acetate-treated platelets with thromin, A23187 and TPA (Fig. 5). Thus, a difference In pHc dependence of the exchangers In resting platelets and in stimulated platelets can not be detected by the measurement of the pHc changes. Differences in the characteristics of the exchangers In resting and stimulated platelets may be clarified by using a method, such as rzNa+ uptake technique, determining the exchange activity directly.
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Since the Nat/H+ exchange is not activated in platelets having high resting-pHc, its activation does not seem to be an indispensable step for Caa+ release in the stimulus-response processes of platelets as described by Siffert and Akkerman (9). Elevation of pHc, however, increases Ca'*-release (9,161 and phospholipase Al activity (7) induced by stimulation, especially with a low dose of agonists. A lowering of the pHc inhibits the metabolism of AA to thromboxan AI in AA-stimulated platelets Phospholipase A1 has pH dependency and increases the (19). sensitivity to Ca'+ with elevation of the pH in vitro (20). Therefore, in platelets having low resting-pHc, elevation of pHc by the Na+/H+ exchanger probably plays a more important role. These findings also suggested that pHc modulates the activity of intracellular reactions, especially when platelets are stimulated with thresholds of agonist doses. Consequently, the stimulationactivated Na+/H+ exchanger is considered to elevate the pHc or prevent its stimulation-induced decrease to maintain the pHc at a constant value and thus modulate intracellular reactions during platelet stimulation.
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EAVOICO, G.B., CRAGOE, E.J.,Jr. and PEINSTEIN, M.B. Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+exchange and its inhibition by amiloride analogs. 2. -Biol. Chem. 261, 13160-13167, 1986.-
2.
LIVNE, A., GRINSTEIN, S. and ROTHSTEIN, A. Characterization of Na+/H' exchange in platelets. Thromb. Haemostas. 58, 971-977, 1987.
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SIPFERT, W., FOX. G., MUCKENHOFF, K. and SCHEID, P. Thrombin stimulates Na+-H+ exchange across the human platelet plasma membrane. --FEBS Lett. 172, 272-274, 1984.
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SIEFFERT, W., SIFFERT, G. and SCHEID. P. Activation of Na+/H+ exchange in human platelets stimulated by thrombin and a phorbol ester. Biochem. J. 241, 301-303, 1987.
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SIFFERT, W., MUCKENHOFF, K. and SCHEID, P. Evidence for a role of Na*/H+exchange in platelets activated with calciumionophore A23187. Biochem. Biophys. Res. Commun. 125, 1123-1128, 1984.
6.
GRINSTEIN, S., COHEN, S. and ROTHSTEIN, A. Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H'antiport. 2. Gen. Physiol. 83, 341-369, 1984.
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SWEATT, J.D., CONNOLLY, T.M., CRAGOE, E.J. and LIMBIRD, L.E. Evidence that Na'/H+ exchange regulates receptor-mediated phospholipase AZ activation in human platelets. 2. Biol. Chem. 261, 8667-8673, 1986.
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8.
BANGA, H.S., SIMONS, E.R., BRASS, L.F. and RITTENHOUSE, S.E. Activation of phospholipases A and C in human platelets exposed to epinephrine: Role of glycoproteins IIb/IIIa and Proc. Natl. Acad. Sci. USA. 83, dual role of epinephrine. ----9197-9201, 1986.
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SIFPERT, W. and AKKERMAN, J.W.N. Activation of sodiumproton exchange is a prerequisite for Ca'+ mobilization in human platelets. Nature, 325, 456-458, 1987.
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SIMPSON, A.W.M. and RINK, T.J. Elevation of pHi is not an essential step in calcium mobilisation in fura-2-loaded human platelets. -FEBS Lett. 222, 144-148, 1987.
11.
HUNYADY, L., SARKADI, B., CRAGOE, E.J.Jr. SPAT, A. and GARDOS, G. Activation of sodium-proton exchange is not a prerequisite for Ca'+mobilization and aggregation in human platelets. -FEBS Lett. 225, 72-76, 1987.
12.
SANCHEZ, A., ALONSO, M.T. and COLLAZOS, J.M. Thrombininduced changes of intracellular [Ca'+l and pH in human platelets. Cytoplasmic alkalinization is not a prerequisite for calcium mobilization. Biochim. Biophys. Acta 938, 497-500, 1988.
13.
ZAVOICO, G.B. and CRAGOE, E.J.,Jr. Ca" mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets. 2. -Biol. Chem-. 263, 9635-9639, 1988.
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FUNDER, J., HERSHCO, L., ROTHSTEIN, A. and LIVNE, A. Na+/H+ exchange and aggregation of human platelets activated by ADP: the exchange is not required for aggregation. Biochim. Biophys. Acta 938, 425-433, 1988.
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VIGNE, P., FRELIN, C. and LAZDUNSKI, M. The Na+/H+antiport is activated by serum and phorbol esters in proliferating myoblasts but not in differentiated myotubes. Properties of the activation process. 2. --Biol. Chem. 260, 8008-8013, 1985.
16.
OGAWA, A., ISHIKAWA, Y. and SASAKAWA, S. Effects of changes in intracellular pH induced by platelet storage and treatment with monovalent cation ionophores on platelet functions. Thromb. Res. 52, 369-379, 1988.
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ARONSON, P.S., NEE, J. and SUHM, M.A. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature, 299, 161-163, 1982.
18.
SIFFERT, W. and AKKERMAN, J.W.N. Protein kinase C enhances Ca'+ mobilization in human platelets by activating Na+/H+ exchange. 2. Biol. Chem. 263, 4223-4227, 1988.
19.
ISHIKAWA, Y., OGAWA, A. and SASAKAWA, S. Effect of cytoplasmic pH on platelet activation by arachidonic acid.
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Thromb. Res. 1989, in press. 20.
BARON, B.M. and LIMBIRD, L.E. Human platelet phospholipase AI activity is responsive in vitro to pH and Cal+variations which parallel those occurring after platelet activation in vivo. Biochim. Biophys. Acta, 971, 103-111, 1988.