Phosphoinositides Are Required for Store-mediated Calcium Entry in Human Platelets*

We have recently observed that small GTP-binding proteins are important for mediation of store-mediated Ca 2 1 entry in human platelets through the reorganization of the actin cytoskeleton. Because it has been shown in platelets and other cells that small GTP-bind-ing proteins regulate the activity of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, whose products, phosphoinositides, play a key role in the reorganization of the actin cytoskeleton, we have investigated the role of these lipid kinases in store-mediated Ca 2 1 entry. Treatment of platelets with LY294002, an inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinases, resulted in a concentration-dependent inhibition of Ca 2 1 entry stimulated by thapsigargin or the physiological agonist, thrombin. In addition, wortmannin, another inhibitor of these kinases, which is structur-ally unrelated to LY294002, significantly reduced store-mediated Ca 2 1 entry. The inhibitory effect of LY294002 was not mediated either by blockage of Ca 2 1 channels or by modification of membrane potential. LY294002 inhibited actin polymerization stimulated by thrombin or thapsigargin. These results indicate that both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase are required for activation of store-mediated Ca 2 1 entry in human platelets and that the mechanism could involve the reorganization of the actin cytoskeleton. platelet-rich plasma was incubated at 37 °C with 2 m M fura-2/AM for 45 min. For co-loading with dimethyl BAPTA, 10 m M dimethyl BAPTA/AM was added for the final 30 min of the fura-2 loading period. Cells were collected by centrifugation and resuspended in HEPES-buffered saline containing (HBS in m M ): 145 NaCl, 10 HEPES, 10 D -glucose, 5 KCl, 1 MgSO 4 , pH 7.45 and supplemented with 0.1% (w/v) BSA and 40 m g/ml apyrase. Measurement of [Ca 2 1 ] i — Fluorescence was recorded from aliquots of stirred platelet suspensions at 37 °C using a Cairn Research Spectrophotometer (Cairn Research Ltd., Kent, UK) with excitation wave- lengths of 340 and 380 nm and emission at 500 nm. Changes in [Ca 2 1 ] i were monitored using the fura-2 340/380 fluorescence ratio and cali- brated according to the method of Grynkiewicz et al. (20). Ca 2 1 influx in TG-induced store-depleted platelets was estimated using the integral of the rise in [Ca 2 1 ] i for 2.5 min after addition of CaCl 2 . Thrombin-evoked Ca 2 1 influx was measured as the integral of the rise in [Ca 2 1 ] i above basal for 2.5 min after addition of thrombin in the presence of external Kinase carry PI3- kinase activity in immunoprecipitates with anti-PI3-kinase p85 antibody complexed with Protein A-agarose The immunoprecipitates incubated 50 m of a buffer 10 m M Tris-HCl, m M m M mg/ml sonicated PtdIns, m M ATP, m M MgCl 2 and 30 m Ci of g - 32 P] ATP for 10 Reactions were by the of m of 6 N HCl and m 3 centrifugation, the lower or-ganic phase was taken for thin layer chromatography on Silica (Merck) O:NH were by autoradiography. The autoradiographs were densitometrically on a Quantimet 500 densitometer PI4-kinase activity was mon- itored in anti-PI4-kinase b antibody immunoprecipitates of human platelets as and g 32 Protein tyrosine phosphorylation was detected by gel electrophoresis and Western blotting with a specific anti-phosphotyrosine antibody (4G10) as described previously Densitometric measurements 500 densitometer (Leica, Milton Keynes, UK), integrated Statistical Analysis— Analysis of statistical significance per- formed using Student’s unpaired t test. multiple comparison, one-way analysis variance combined tests used. n M z 2093 n M z in LY294002-treated the presence findings are in agreement of and

Store-mediated Ca 2ϩ entry (SMCE) 1 is a mechanism present in many cell types; however, the intracellular processes underlying SMCE remain unclear (1). Several hypotheses have considered both direct and indirect coupling mechanisms (2). Indirect coupling assumes the existence of a diffusible messenger generated by the intracellular Ca 2ϩ stores; in contrast, direct coupling models propose a physical interaction between the endoplasmic reticulum (ER) and the plasma membrane (PM; Ref. 2). Recently a new model for SMCE has been proposed in several different cell types. This involves a physical but reversible interaction between the ER and the PM that may require translocation of portions of the ER toward the PM and mechanical support provided by the actin cytoskeleton (3,4). In support of this hypothesis, small GTP-binding proteins, which modulate vesicular transport through the reorganization of the actin cytoskeleton and the actin cytoskeleton itself, have been suggested to be important for SMCE in different cell types (5)(6)(7) including platelets (8,9).
Here we report for the first time the involvement of PI3-and PI4-kinases in SMCE. In addition, the possible involvement of the actin cytoskeleton in phosphoinositide-mediated SMCE was investigated in human platelets.
Measurement of [Ca 2ϩ ] i -Fluorescence was recorded from aliquots of stirred platelet suspensions at 37°C using a Cairn Research Spectrophotometer (Cairn Research Ltd., Kent, UK) with excitation wavelengths of 340 and 380 nm and emission at 500 nm. Changes in [Ca 2ϩ ] i were monitored using the fura-2 340/380 fluorescence ratio and calibrated according to the method of Grynkiewicz et al. (20). Ca 2ϩ influx in TG-induced store-depleted platelets was estimated using the integral of the rise in [Ca 2ϩ ] i for 2.5 min after addition of CaCl 2 . Thrombin-evoked Ca 2ϩ influx was measured as the integral of the rise in [Ca 2ϩ ] i above basal for 2.5 min after addition of thrombin in the presence of external * This work was supported in part by Grant 051560 from The Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Supported by a Grant of the Junta de Extremadura-Consejería de Educación y Juventud and Fondo Social Europeo, Spain.
Phosphatidylinositol Kinase Assay-PI3-kinase activity was carry out essentially as described by Way and Mooney (21). Briefly, PI3kinase activity was measured in immunoprecipitates with anti-PI3kinase p85 antibody complexed with Protein A-agarose beads. The immunoprecipitates were incubated in 50 l of a buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.2 mg/ml sonicated PtdIns, 50 M ATP, 20 mM MgCl 2 , and 30 Ci of [␥-32 P] ATP at 37°C for 10 min. Reactions were stopped by the addition of 20 l of 6 N HCl and 160 l of CHCl 3 :MeOH (1:1). Following centrifugation, the lower organic phase was taken for thin layer chromatography on Silica Gel 60 plates (Merck) to be developed in CHCl 3 :MeOH:H 2 O:NH 4 OH (60:47: 11.3:2). Radioactive spots were visualized by autoradiography. The autoradiographs were densitometrically scanned on a Quantimet 500 densitometer (Leica, Milton Keynes, UK). PI4-kinase activity was monitored in anti-PI4-kinase ␤ antibody immunoprecipitates of human platelets as described by Meyers and Cantley (22). 32 P-Phospholipid Labeling-Human platelets were labeled with [␥-32 P]phosphate as described previously (12). 32 P-Labeled platelets were preincubated at 37°C for 30 min with vehicle or LY294002 followed by treatment with TG. Lipids were extracted, separated, and quantified as described above.
Measurement of F-actin Content-The cellular content of F-actin was analyzed by staining with fluorescein isothiocyanate-labeled phalloidin as described previously (11). The fluorescence of the different samples was determined in a Perkin-Elmer fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT). Samples were excited at 496 nm, and emission was at 516 nm.
Protein Tyrosine Phosphorylation-Protein tyrosine phosphorylation was detected by gel electrophoresis and Western blotting with a specific anti-phosphotyrosine antibody (4G10) as described previously (19). Densitometric measurements were made using a Quantimet 500 densitometer (Leica, Milton Keynes, UK), and the integrated optical density of the entire lane was estimated.
Statistical Analysis-Analysis of statistical significance was performed using Student's unpaired t test. For multiple comparison, oneway analysis of variance combined with the Dunnett tests was used.

RESULTS AND DISCUSSION
LY294002 Inhibits PI3-and PI4-kinase Activity and the Synthesis of PtdIns Phosphate-The activity of PI3-and PI4-kinase ␤ was determined by immunocomplex assay as described under "Experimental Procedures." The effect of LY294002 on PI3-and PI4-kinase ␤ activity immunocomplexed from human platelets is shown in Fig. 1A. LY294002 inhibited the activity of PI3-and PI4-kinase ␤ in a concentration-dependent manner, with an IC 50 of 1.5 and 34.4 M for PI3-kinase and PI4-kinase ␤ activity, respectively. These results are similar to those reported by Effect of LY294002 or Wortmannin on TG-evoked SMCE-In a Ca 2ϩ -free medium TG evoked a prolonged elevation of [Ca 2ϩ ] i in platelets due to release of Ca 2ϩ from the intracellular stores. Subsequent addition of Ca 2ϩ (final concentration 300 M) to the external medium resulted in a sustained increase in [Ca 2ϩ ] i indicative of SMCE ( Fig. 2A). Pretreatment of human platelets for 30 min at 37°C with different concentrations of LY294002 decreased TG-evoked Ca 2ϩ entry in a concentration-dependent manner (Fig. 2, A and B). Treatment of platelets with low concentrations of LY294002 (10 M), conditions under which PI3-kinase activity is inhibited in vitro (Fig. 1), slightly reduced TG-evoked SMCE by 31 Ϯ 4% ( Fig. 2B; p Ͻ 0.05). However, pretreatment of platelets with higher concentrations of LY294002 (100 M), conditions under which PI4kinase activity is almost completely inhibited in vitro (Fig. 1), significantly reduced TG-induced SMCE to 17 Ϯ 4% of control ( Fig. 2B; p Ͻ 0.001).
Consistent with the above, treatment of human platelets for 10 min with 10 nM WT, conditions under which PI3-kinase activity has been shown to be inhibited in several cell types (24,25) including platelets (12,13), slightly reduced TG-induced SMCE by 18 Ϯ 4% ( Fig. 2C; p Ͻ 0.05). In addition, treatment of platelets for 10 min with 1 M WT, a concentration reported to inhibit PI4-kinase activity (22,24,25), significantly reduced TG-evoked SMCE by 81 Ϯ 2% ( Fig. 2C; p Ͻ 0.001). LY294002treated cells retained their ability to respond to Ca 2ϩ -mobilizing agents such as TG (Fig. 2A). A similar effect was observed with WT (not shown). These findings indicate that treatment of

Phosphoinositides and Calcium Entry 9111
platelets with these agents did not affect their ability to store Ca 2ϩ in intracellular compartments.

LY294002 Reduces Thrombin-evoked [Ca 2ϩ ] i Elevation-
Thrombin is a physiological agonist that stimulates a large number of processes in platelets such as calcium mobilization and activation of PI3-and PI4-kinases (19,26). Treatment of human platelets for 30 min at 37°C with LY294002 at concentrations of 10 or 100 M reduced the rise in [Ca 2ϩ ] i evoked by thrombin (0.1 unit/ml) in a medium containing 1 mM Ca 2ϩ (Fig. 3, A and C). The initial peak [Ca 2ϩ ] i elevation above basal after agonist stimulation was significantly reduced from 349 Ϯ 23 to 271 Ϯ 8 or 166 Ϯ 9 nM after treatment with 10 or 100 M LY294002, respectively (p Ͻ 0.05). If we consider the entry of Ca 2ϩ stimulated by thrombin (see "Experimental Procedures"), LY294002 significantly reduced thrombin-evoked Ca 2ϩ entry by 30 Ϯ 5 and 75 Ϯ 2% at concentrations 10 and 100 M, respectively (p Ͻ 0.01).
In the absence of external Ca 2ϩ (100 M EGTA added) 10 M LY294002 was without effect on thrombin-induced rise in [Ca 2ϩ ] i . The initial peak elevation in [Ca 2ϩ ] i above basal after agonist stimulation was 129 Ϯ 8 nM in control cells and 136 Ϯ 6 nM in LY294002-treated cells (Fig. 3B). However, interestingly treatment of platelets with 100 M LY294002 reduced thrombin-induced release of Ca 2ϩ from the intracellular stores by 26 Ϯ 5% (Fig. 3D; p Ͻ 0.01) indicating that 100 M but not 10 M concentration of the inhibitor affects PI4-kinase activity, which is responsible for regulating the synthesis of agonistsensitive pools of phosphoinositides, thus limiting the amount of PtdIns-4,5-P 2 available to phospholipase C for the generation of the Ca 2ϩ releasing messenger, Ins(1,4,5)P 3 .
Although our results suggest that both kinases may be implicated in the activation of SMCE, the small effect observed after treatment of platelets with LY294002 or WT at concentrations that specifically inhibit PI3-kinase suggests that PI4kinase rather than PI3-kinase is in the main responsible for the activation of SMCE or PI4-kinase activity may impair the inhibition of PI3-kinase by phosphorylating PtdIns-3-P molecules remaining in the cell. To our knowledge this is the first time that PI3-and PI4-kinases have been shown to be involved in SMCE.
Effect of LY294002 on Tyrosine Phosphorylation Induced by Depletion of the Intracellular Ca 2ϩ Stores-We have previously reported a general inhibitory effect of WT on tyrosine phosphorylation in human platelets (27), which has been extensively reported to be required for SMCE (e.g. Ref. 19). Hence we have now examined the effect of LY294002 on store depletion-evoked tyrosine phosphorylation in these cells. Platelets heavily loaded with the Ca 2ϩ chelator dimethyl-BAPTA were used for this study so as to eliminate Ca 2ϩ -but not store depletiondependent tyrosine phosphorylation (28). Dimethyl BAPTAloaded platelets were incubated for 30 min at 37°C in the presence of LY294002 or the vehicle, and Ca 2ϩ stores were depleted using TG (250 nM) and ionomycin (50 nM). In each experiment we checked that the rise in [Ca 2ϩ ] i evoked by TG and ionomycin was abolished by BAPTA loading (data not shown). Samples for protein phosphorylation analysis were taken from the spectrophotometer cuvette 10 s prior, and 45, 170, and 300 s after the addition of TG and ionomycin. As shown in Table I, LY294002 did not significantly modify phosphotyrosine levels at the highest concentration used in this

TABLE I
Effects of LY294002 on the phosphotyrosine content of store-depleted BAPTA-loaded platelets Dimethyl-BAPTA-loaded platelets were preincubated for 30 min at 37°C with either 100 M LY294002 or Me 2 SO 4 (control) and then were treated with 250 nM TG and 50 nM ionomycin to deplete the intracellular Ca 2ϩ stores. Samples were taken from the platelet suspension at 10 s before and 45, 170, and 300 s after the addition of TG and ionomycin. Platelet proteins were analyzed by 10% SDS-polyacrylamide gel electrophoresis and subsequent Western blot with a specific antiphosphotyrosine antibody, and the presence of phosphotyrosine residues was quantified by densitometry in Western blots as described. The data represent the integrated optical density for entire lanes under each condition. Results are expressed as -fold increase (mean Ϯ S.E.) over the integrated optical density of platelet proteins before Ca 2ϩ store depletion. Phosphoinositides and Calcium Entry 9112 study. The data presented here demonstrated that, in contrast to the effect reported for WT, preincubation for 30 min with the highest concentration of LY294002 used in this study did not have a general inhibitory effect on tyrosine kinase activity stimulated by depletion of intracellular Ca 2ϩ stores.
To assess whether the inhibitory effect of LY294002 on SMCE could be mediated by Ca 2ϩ chelation or Ca 2ϩ channel blockage we examined the effect of LY294002 after initiation of SMCE. As shown in Fig. 4, addition of 100 M LY294002 2 min after the addition of Ca 2ϩ to store-depleted cells did not modify TG-evoked Ca 2ϩ entry for up to 30 min, the preincubation time used in this study. In contrast, addition of 100 M lanthanum, a nonspecific cation channel blocker, caused a rapid decrease in [Ca 2ϩ ] i over the same time course, indicating that Ca 2ϩ entry had been inhibited (Fig. 4). The lack of effect of LY294002 after SMCE has been stimulated using TG indicate that the inhibition of Ca 2ϩ entry by LY294002 is not due to nonspecific effects as a chelator of Ca 2ϩ or as a Ca 2ϩ entry channel blocker. In addition, our results indicate that PI3-and PI4-kinase activity is required for the activation of SMCE but not for its maintenance in human platelets.
Since depolarization of the membrane potential has been shown to reduce the driving force for Ca 2ϩ entry we also investigated whether the inhibitory effects of LY294002 could be attributed to changes in membrane potential by studying the effect of this inhibitor on SMCE in the presence of the valinomycin, which stabilizes the platelet membrane potential close to the K ϩ equilibrium potential (29). Treatment of platelets with 100 M LY294002 inhibited TG-induced SMCE to the same extent in the presence or absence of valinomycin (3 M) (not shown). Ca 2ϩ influx was 57278 Ϯ 9742 nM⅐s under control conditions and 7043 Ϯ 1250 nM⅐s in LY294002-treated cells in the absence of valinomycin (87% inhibition) and 70372 Ϯ 8058 nM⅐s in control and 11488 Ϯ 2093 nM⅐s in LY294002-treated cells in the presence of valinomycin (84% inhibition), respectively (n ϭ 3). These findings indicate that the effects of LY294002 are not due to a reduction in the membrane potential. These results together with others presented in this paper indicate that the effect of LY294002 on SMCE is more likely to be explained by inhibition of PI3-and PI4-kinases. These findings are in agreement with previous studies reporting the high specificity of LY294002 as an inhibitor of PI3-and PI4-kinase activity (22).
LY294002 Inhibits Actin Polymerization in Platelets-The actin cytoskeleton has been shown to play a key role in the activation and maintenance of SMCE in platelets (8,9) and other cells (3,7), and phosphoinositides are required for actin polymerization (10,11). Hence we have now investigated the effect of different concentrations of LY294002 on actin filament formation in platelets to assess whether the involvement of phosphoinositides on SMCE activation could be in the reorganization of the actin filament network. Platelets were incubated for 30 min in the absence or presence of LY294002 at concentrations 10 or 100 M. Cells were then treated either with 200 nM TG before addition of Ca 2ϩ (300 M) or with thrombin (0.1 unit/ml) in a medium containing 1 mM Ca 2ϩ , and F-actin content was determined. Treatment of human platelets with LY294002 significantly reduced thrombin or TG-evoked increase in F-actin content by 52 Ϯ 5 and 56 Ϯ 5%, respectively, at a concentration of 10 M and by 81 Ϯ 2 and 79 Ϯ 5%, respectively, when 100 M LY294002 was used ( Fig. 5; p Ͻ 0.001). Therefore, LY294002 reduced actin polymerization induced by TG or thrombin at concentrations that inhibit both PI3-and PI4-kinases. Our observations are in agreement with our previous studies reporting that actin polymerization is required for the activation and maintenance of SMCE in human platelets (8,9). Treatment of platelets with LY294002 had no effect on basal levels of F-actin (data not shown) indicating that either treadmilling is very slow in human platelets or phosphoinositides are not important for the maintenance of the actin network in resting platelets.
In conclusion, the results of the current study demonstrate that the synthesis of phosphoinositides is required for the activation of SMCE in human platelets. The activity of PI3kinase and PI4-kinase may represent one of the factors involved in the regulation of the phosphoinositide pool required for the activation of SMCE in these cells, probably through the reorganization of the actin cytoskeleton. These data are thus compatible with a physical coupling model for SMCE, which requires actin polymerization for both translocation of the ER toward the PM for initiation of Ca 2ϩ entry and cytoskeletal support for its maintenance (8).