Phosphatidylinositol 4,5-Bisphosphate Mediates Ca2+-induced Platelet alpha -Granule Secretion

EVIDENCE FOR TYPE II PHOSPHATIDYLINOSITOL 5-PHOSPHATE 4-KINASE FUNCTION*

Nataliya Rozenvayn and Robert FlaumenhaftDagger

From the Division of Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

Received for publication, September 7, 2000, and in revised form, April 6, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To understand the molecular basis of granule release from platelets, we examined the role of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in alpha -granule secretion. Streptolysin O-permeabilized platelets synthesized PtdIns(4,5)P2 when incubated in the presence of ATP. Incubation of streptolysin O-permeabilized platelets with phosphatidylinositol-specific phospholipase C reduced PtdIns(4,5)P2 levels and resulted in a dose- and time-dependent inhibition of Ca2+-induced alpha -granule secretion. Exogenously added PtdIns(4,5)P2 inhibited alpha -granule secretion, with 80% inhibition at 50 µM PtdIns(4,5)P2. Nanomolar concentrations of wortmannin, 33.3 µM LY294002, and antibodies directed against PtdIns 3-kinase did not inhibit Ca2+-induced alpha -granule secretion, suggesting that PtdIns 3-kinase is not involved in alpha -granule secretion. However, micromolar concentrations of wortmannin inhibited both PtdIns(4,5)P2 synthesis and alpha -granule secretion by ~50%. Antibodies directed against type II phosphatidylinositol-phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) also inhibited both PtdIns(4,5)P2 synthesis and Ca2+-induced alpha -granule secretion by ~50%. These antibodies inhibited alpha -granule secretion only when added prior to ATP exposure and not when added following ATP exposure, prior to Ca2+-mediated triggering. The inhibitory effects of micromolar wortmannin and anti-type II phosphatidylinositol-phosphate kinase antibodies were additive. These results show that PtdIns(4,5)P2 mediates platelet alpha -granule secretion and that PtdIns(4,5)P2 synthesis required for Ca2+-induced alpha -granule secretion involves the type II phosphatidylinositol 5-phosphate 4-kinase-dependent pathway.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha -Granules are the dominant platelet secretory granules. They contain many components that have been implicated in thrombosis and atherosclerosis, including adhesion molecules, coagulation factors, soluble mediators of inflammation, and growth factors (1). The molecular mechanisms that mediate the release of these components are not well defined. This secretory process is tightly controlled to prevent unregulated release of thrombogenic factors. In many respects, platelets represent a unique secretory system. They are anucleate, contain an open canalicular system that undergoes eversion following platelet activation (2), and demonstrate homotypic granule fusion (3). Ultrastructural studies have shown that the alpha -granules of platelets are secreted primarily via fusion with the surface-connected open canalicular system (1) following apparent centralization of granules and microtubule reorganization. Such observations have lead to speculation that cytoskeletal reorganization is responsible for alpha -granule release. However, inhibition of actin polymerization (4) or microtubule organization (5, 6) does not inhibit granule secretion. Furthermore, granule secretion and shape change can be dissociated under several experimental conditions (7-9). More recently, SNARE1 proteins have been demonstrated in platelets (10-12) and shown to mediate platelet alpha -granule secretion (12-15). Purified SNARE proteins are capable of fusing lipid membranes in vitro (16, 17). However, permeabilized cell systems require molecules other than SNARE proteins to secrete granules. Furthermore, regulated secretion necessitates that the SNARE protein apparatus respond to activation-dependent signals (18). Thus, an understanding of the molecular mechanisms of platelet granule secretion requires the identification of molecules that are both modified upon platelet activation and capable of interacting directly with the secretory machinery.

Phospholipid metabolism has long been regarded as an essential component of regulated secretion in platelets. Initial studies performed in intact (19) and electropermeabilized (20) platelets indicated a role for the products of phosphatidylinositol-specific PLC, diacylglycerol and inositol 1,4,5-trisphosphate, in stimulating granule secretion (21). However, both dense and alpha -granule secretion can be stimulated in the absence of PLC activation, suggesting that PLC activity is not essential to platelet granule secretion (22-24). Subsequent studies in Ca2+-independent permeabilized secretory systems showed that the generation of phosphatidic acid by phospholipase D correlates closely with secretion (23, 24). Inhibition of phospholipase D, however, fails to block Ca2+-independent dense granule secretion, and phosphatidic acid itself cannot stimulate dense granule secretion (25). These observations support a modulatory role of phospholipase D in platelet granule secretion. Thus, no product of phospholipid metabolism has been demonstrated to be fusogenic in platelets (25), and the role of phospholipid metabolism in platelet membrane fusion remains to be defined.

Phosphoinositide phosphorylation has been invoked in vesicle fusion models and shown to mediate granule secretion. Initial observations in chromaffin cells demonstrated that the maintenance of polyphosphoinositides is crucial for vesicle secretion (26). Subsequent studies revealed a PtdIns 4-kinase activity associated with granules in chromaffin, mast, and pancreatic beta -cells (27). In chromaffin cells, this PtdIns 4-kinase activity correlates with granule secretion (28). In PC12 cells, phosphatidylinositol transfer protein was determined to be one of three cytosolic factors required to restore Ca2+-induced secretion from semi-intact PC12 cells (29). Type I PIPK is a second cytosolic factor necessary to reconstitute secretion (30). Subsequent to these studies, phosphatidylinositol transfer protein was found to reconstitute Ca2+-induced, GTPgamma S-dependent granule secretion from SL-O-permeabilized HL-60 cells (31). In RBL-2H3 mast cells, ADP-ribosylation factor-1 was demonstrated to stimulate exocytosis via PtdIns(4,5)P2 synthesis (32). These data suggest a central role for PtdIns(4,5)P2 in regulated secretion.

Although the role of PtdIns(4,5)P2 itself in platelet granule secretion has not previously been evaluated, PtdIns(4,5)P2 metabolism has been studied in platelets, and PtdIns(4,5)P2 has been shown to mediate several other platelet processes. PtdIns(4,5)P2 is involved in uncapping of F-actin barbed ends, thereby contributing to platelet shape change (33). PtdIns(4,5)P2 is also thought to mediate platelet spreading (34). Upon platelet activation with thrombin (35, 36) or thrombin receptor agonist peptides (33), PtdIns(4,5)P2 increases 10-40% above base-line levels. The generation of PtdIns(4,5)P2 is presumed to be mediated by up-regulation of PtdIns kinases. However, the synthetic pathway by which PtdIns(4,5)P2 is synthesized in platelets is not well established. Synthesis of PtdIns(4,5)P2 via progressive phosphorylation of PtdIns by PtdIns 4-kinase followed by PtdIns-4-phosphate 5-kinase represents the dominant pathway in most cell types. However, platelet PtdIns 5-phosphate levels increase upon exposure to thrombin (37). In addition, type II PIPK is abundant in platelets (38) and undergoes aggregation-dependent translocation to the platelet cytoskeleton (39). These observations raise the possibility that PtdIns(4,5)P2 can be synthesized via a PtdIns-5-phosphate 4-kinase-dependent pathway. In this study, we demonstrate that PtdIns(4,5)P2 mediates platelet alpha -granule secretion and that PtdIns(4,5)P2 synthesis required for alpha -granule secretion involves the type II PtdIns 5-phosphate 4-kinase-dependent pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals and Reagents-- All buffer constituents, solvents, ATP, CaCl2, PtdIns, PtdIns 4-phosphate, PtdIns(4,5)P2, and PtdIns-specific PLC (from Bacillus cereus) were purchased from Sigma. Sepharose 2B was obtained from Amersham Pharmacia Biotech. Reduced SL-O was purchased from Corgenix (Peterborough, England). Wortmannin and LY294002 were purchased from Calbiochem. Phycoerythrin-conjugated anti-P-selectin antibody AC1.2 was purchased from Becton Dickinson (San Jose, CA). [32P]Orthophosphoric acid was obtained from PerkinElmer Life Sciences. Silica Gel G TLC plates were obtained from Whatman Ltd. (Kent, England). GST-tagged type II PIPK was generously provided by Dr. Lucia Rameh. All solutions were prepared using water purified by reverse-phase osmosis on a Millipore Milli-Q purification water system.

Antibodies-- Affinity-purified mouse monoclonal antibodies to PtdIns 3-kinase p85alpha (clone 8-2D-4D) were obtained from Lab Vision (Fremont, CA). Affinity-purified rabbit anti-peptide polyclonal antibodies to type II PIPK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-amino-terminal end antibody was raised against a peptide consisting of the 19 amino-terminal amino acids of type IIalpha PIPK. The anti-carboxyl-terminal end antibody was raised against a peptide consisting of the 18 carboxyl-terminal amino acids of type IIalpha PIPK.

Platelet Preparation-- Blood from healthy donors who had not ingested aspirin in the 2 weeks prior to donation was collected by venipuncture into 0.4% sodium citrate. Citrate-anticoagulated blood was centrifuged at 200 × g for 20 min to prepare platelet-rich plasma. Platelets were then purified from platelet-rich plasma by gel filtration using a Sepharose 2B column equilibrated in PIPES/EGTA buffer (25 mM PIPES, 2 mM EGTA, 137 mM KCl, 4 mM NaCl, and 0.1% glucose, pH 6.4). Final gel-filtered platelet concentrations were 1-2 × 108 platelets/ml.

Permeabilization of Platelets-- Platelets were permeabilized using reduced SL-O. The ability of each batch of SL-O to permeabilize platelets was tested by analyzing for incorporation of fluorescein isothiocyanate-dextran sulfates by flow cytometry as described previously (12, 40). Briefly, gel-filtered platelets (20 µl) were incubated with 25 µM fluorescein isothiocyanate-dextran sulfate of various molecular masses in the presence or absence of SL-O for 15 min. Platelets were subsequently analyzed for fluorescence by flow cytometry. An increase in fluorescence in the samples exposed to SL-O compared with non-permeabilized samples confirmed permeabilization.

Analysis of P-selectin Surface Expression-- For analysis of P-selectin surface expression from permeabilized platelets, 20 µl of gel-filtered platelets (1-2 × 108/ml) in the indicated concentration of MgATP were incubated with the concentrations of reduced SL-O indicated in the figure legends. Samples were adjusted to pH 6.9 immediately following permeabilization. The timing of addition of inhibitors varied according to the inhibitor that was being evaluated and is indicated in the figure legends. Following the incubation with inhibitor, CaCl2 was added to the reaction mixture. The amount of CaCl2 required to give a free Ca2+ concentration of 10 µM in the presence of 2 mM EGTA at pH 6.9 was calculated for each condition using a computer program (gift from Dr. P. J. Padfield) based on the algorithms described by Fabiato and Fabiato (41). Following an additional incubation after the addition of Ca2+, 10 µl of reaction mixture were transferred to 5 µl of phycoerythrin-conjugated anti-P-selectin antibody AC1.2. Phosphate-buffered saline (500 µl) was added to the sample after a 20-min incubation, and the platelets were analyzed immediately by flow cytometry as described below.

Immunoblot Analysis-- Gel-filtered platelets (1-2 × 107/ml) or GST-tagged PIPK was diluted in sample buffer (62.5 mM Tris-HCl, 2% SDS, 0.5% beta -mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue) at 95 °C for 5 min. Proteins were then separated by SDS-polyacrylamide gel electrophoresis on 12% gels. Immunoblotting was performed using anti-type II PIPK antibodies directed against the amino- and carboxyl-terminal ends of the alpha -isoform of human type II PIPK and visualized using enhanced chemiluminescence.

Analysis of Platelet Phosphoinositides-- Gel-filtered platelets (350 µl/sample) were incubated in the presence of 2 mCi/ml [32P]orthophosphoric acid at 37 °C for 2 h. Unbound 32P was separated from platelets by sequential centrifugation. Platelets were then permeabilized with the indicated concentrations of SL-O in the presence or absence of ATP. Samples were adjusted to pH 6.9 immediately following permeabilization. The timing of addition of inhibitors varied according to the inhibitor and is indicated in the figure legends. Platelets were subsequently solubilized in 250 µl of a 20:40:1 solution of chloroform, methanol, and 12 N HCl and mixed vigorously. Chloroform (75 µl) was added to this solution. The solution was mixed vigorously, and its phases were separated at 1300 × g for 10 min at 4 °C. The upper phase was discarded, and the lower phase was washed three times with 625 µl of the 20:40:1 solution of chloroform, methanol, and 12 N HCl. Each extract (5 µl) was applied to one lane of a 20 × 20-cm Silica Gel G plate previously soaked in 1% potassium oxalate for 30 min and then baked for 5 min at 100 °C. Phospholipids were separated by chromatography in 1-propanol and 2 M acetic acid (65:35). The location of PtdIns 4-phosphate and PtdIns(4,5)P2 was determined by applying 5 µg of known standards to each lane. Standards were detected by staining in a saturated iodine tank. The radioactivity on the plates was detected using a Bio-Rad GS-525 molecular imaging system. Phosphoinositide levels were quantified both by calculating the pixel density of bands comigrating with PtdIns standards using Molecular Analyst software (Bio-Rad) and by extracting the radioactivity comigrating with the PtdIns standards from the plate and quantifying radioactivity using a Tri-Carb 2100TR liquid scintillation analyzer (Packard Instrument Co.). Under the conditions of our assay, a pixel density of 1 corresponds to 10 cpm.

Flow Cytometry-- Flow cytometry was performed on gel-filtered platelet samples using a Becton Dickinson FACSCalibur flow cytometer. Fluorescent channels were set at logarithmic gain. 10,000 particles were acquired for each sample. A 530/30-nm band-pass filter was used for FL-2 fluorescence. Phycoerythrin was measured in the FL-2 channel. Data were analyzed using CellQuest software on a Macintosh Power PC.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SL-O-permeabilized Platelets Synthesize PtdIns(4,5)P2-- To study the molecular mechanisms of platelet alpha -granule secretion, we have developed and characterized an SL-O-permeabilized, Ca2+-induced alpha -granule secretory system (12). In this system, exposure of platelets to SL-O permits the entry of molecules as large as 260 kDa (12). In the presence of 5 mM ATP, 96 ± 1% of platelets exposed to SL-O expressed P-selectin on their surface in response to 10 µM Ca2+. In contrast, non-permeabilized platelets expressed almost no P-selectin on their surface in response to ATP and Ca2+ (Fig. 1) (12). These observations demonstrate that >95% of platelets were permeabilized following incubation with SL-O. Multiple Ca2+-induced permeabilized secretory systems, including permeabilized platelet models (42, 43), have demonstrated a requirement for ATP exposure prior to Ca2+-induced triggering of granule secretion. Many permeabilized secretory systems also require cytosol to reconstitute significant secretion (44). A role for PtdIns(4,5)P2 synthesis in PC12 cell granule secretion was demonstrated when type I PIPK and PtdIns transfer protein were identified as proteins within cytosol responsible for reconstituting ATP-mediated priming of granule secretion (29, 30). In contrast, cytosol is not required for Ca2+-mediated secretion of alpha -granules from SL-O-permeabilized platelets (12). However, alpha -granule secretion from SL-O-permeabilized platelets remained ATP-dependent (Fig. 1). ATP did not support alpha -granule secretion from non-permeabilized platelets or under conditions in which the free Ca2+ concentration was maintained at pCa2+ < 8 with 2 mM EGTA (Fig. 1B). The ability of ATP to support alpha -granule secretion was dose-dependent with an EC50 of ~1 mM. ADP neither supported alpha -granule secretion from SL-O-permeabilized platelets nor augmented Ca2+-induced alpha -granule secretion in the presence of submaximal concentrations of ATP (Fig. 1C). These data demonstrate that Ca2+-induced alpha -granule secretion in this system requires permeabilization with SL-O and exposure to ATP.


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Fig. 1.   Effect of ATP on alpha -granule secretion from SL-O-permeabilized platelets. A, gel-filtered platelets (20 µl/sample) in PIPES/EGTA buffer supplemented with 5 mM MgCl2 were permeabilized with 4 units/ml SL-O in the presence or absence of 5 mM ATP at pH 6.9 for 10 min. Platelets were then exposed to 10 µM Ca2+ for 10 min. P-selectin surface expression was assayed by incubating the sample (10 µl) with phycoerythrin-conjugated anti-P-selectin antibody AC1.2 (5 µl) for 20 min. Phosphate-buffered saline (500 µl) was added to the sample, and the platelets were analyzed immediately by flow cytometry as described under "Experiment Procedures." Histograms represent the distribution of the relative fluorescence of 10,000 platelets. B, gel-filtered platelets in PIPES/EGTA buffer supplemented with 5 mM MgCl2 were incubated in the presence or absence of 5 mM ATP as indicated. Platelets were then incubated in the presence or absence of 4 units/ml SL-O at pH 6.9 for 10 min and subsequently exposed to buffer or 10 µM Ca2+ for 10 min. Platelets were assayed for P-selectin expression by flow cytometry. Error bars represent the S.E. of three to seven independent experiments. C, gel-filtered platelets in PIPES/EGTA buffer supplemented with 5 mM MgCl2 were permeabilized with 4 units/ml SL-O in the presence of the indicated concentrations of ATP at pH 6.9 for 10 min in the presence (open circle ) or absence () of 500 µM ADP. Platelets were then exposed to 10 µM Ca2+ for 10 min and assayed for P-selectin expression by flow cytometry. Error bars represent the S.E. of six independent experiments.

The observation that ATP, but not platelet cytosol, is required for Ca2+-induced alpha -granule secretion raises the question of whether SL-O-permeabilized platelets are able to synthesize PtdIns(4,5)P2 upon exposure to ATP in the absence of added cytosol. To address this question, platelets were radiolabeled with [32P]orthophosphate, incubated in the presence or absence of ATP, and analyzed for PtdIns(4,5)P2 levels by TLC. PtdIns(4,5)P2 levels were markedly increased in SL-O-permeabilized platelets incubated in the presence of ATP compared with levels in platelets incubated in the absence of ATP (Fig. 2A). The contribution of radioactivity from the 4 ± 1% of platelets that were not permeabilized upon exposure to SL-O was calculated to constitute ~1% of the total radioactivity in the PtdIns(4,5)P2 band. We next performed a time course study to determine whether the ability to secrete alpha -granules in response to Ca2+ exposure is temporally correlated with PtdIns(4,5)P2 synthesis. PtdIns(4,5)P2 synthesis and the degree of alpha -granule secretion in response to Ca2+ increased in parallel in SL-O-permeabilized platelets over the first 30 min following exposure to ATP (Fig. 2B). Thus, SL-O-permeabilized platelets are capable of synthesizing PtdIns(4,5)P2 in response to ATP without the addition of cytosol, and PtdIns(4,5)P2 synthesis correlates temporally with the ability to secrete alpha -granules in response to Ca2+ exposure.


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Fig. 2.   Effect of ATP on PtdIns(4,5)P2 synthesis in SL-O-permeabilized platelets. A, gel-filtered platelets (350 µl/sample) in PIPES/EGTA buffer supplemented with 5 mM MgCl2 were labeled with 2 mCi/ml [32P]orthophosphoric acid at 37 °C for 2 h. 32P-Labeled platelets were then permeabilized with 3 units/ml SL-O in the presence or absence of 5 mM ATP at pH 6.9 for 30 min. The lipids were extracted, and PtdIns(4,5)P2 levels were analyzed by TLC as described under "Experimental Procedures." Error bars represent the S.E. of six samples. B, to assess the time course of PtdIns(4,5)P2 synthesis, gel-filtered platelets were labeled with [32P]orthophosphoric acid and permeabilized with 2 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for the indicated amounts of time. The lipids were then extracted, and PtdIns(4,5)P2 levels were analyzed by TLC (). To assess the time course of P-selectin surface expression, gel-filtered platelets were permeabilized with 2 units/ml SL-O in the presence of 5 mM MgATP, 10 µM Ca2+, and phycoerythrin-conjugated anti-P-selectin antibody (5 µl) at pH 6.9 for the indicated amounts of time. Samples were then diluted in phosphate-buffered saline (500 µl) and analyzed immediately by flow cytometry (diamond ). Data are expressed as the percentage of P-selectin expression compared with a sample permeabilized in the presence of 5 mM MgATP and 10 µM Ca2+ for 30 min and subsequently incubated with phycoerythrin-conjugated anti-P-selectin antibody for the indicated amounts of time. Error bars represent the S.E. of four independent experiments.

Effect of PtdIns-specific PLC on Ca2+-induced alpha -Granule Secretion-- To determine whether PtdIns(4,5)P2 mediates Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets, we hydrolyzed endogenous platelet PtdIns(4,5)P2 using PtdIns-specific PLC. PtdIns-specific PLC reduced endogenous PtdIns(4,5)P2 levels in the SL-O-permeabilized platelets by ~75% (Fig. 3A). However, the use of PtdIns-specific PLC in secretory assays is complicated by the fact that the by-products of PtdIns-specific PLC activity, diacylglycerol and inositol 1,4,5-trisphosphate, are themselves capable of influencing secretion from permeabilized platelets (19). Diacylglycerol acts primarily via activation of protein kinase C (21). Sloan and Haslam (45) have shown that protein kinase C diffuses out of platelets following exposure to SL-O. Consistent with these observations, we found that stimulation of P-selectin surface expression by phorbol 12-myristate 13-acetate was reduced by >90% in SL-O-permeabilized platelets compared with non-permeabilized platelets (data not shown). Thus, permeabilized platelets are relatively insensitive to diacylglycerol because of protein kinase C leakage. Inositol 1,4,5-trisphosphate is not likely to stimulate secretion in this system because Ca2+ chelation by EGTA blocks the increase in [Ca2+]i that is elicited by inositol 1,4,5-trisphosphate in unchelated systems. In the secretion experiments, platelets were permeabilized with SL-O for 15 min prior to exposure to PtdIns-specific PLC. Platelets were then incubated in the presence or absence of PtdIns-specific PLC for 15 min prior to Ca2+ exposure. Ca2+-induced alpha -granule secretion was inhibited in permeabilized platelets exposed to PtdIns-specific PLC (Fig. 3B). Platelets incubated with PtdIns-specific PLC in the absence of Ca2+ exposure demonstrated a small degree of secretion under these conditions (8 ± 2% P-selectin expression compared with samples incubated with buffer and subsequently exposed to Ca2+) that might result from the generation of diacylglycerol. Heat-denatured PtdIns-specific PLC had no effect on Ca2+-induced alpha -granule secretion. PtdIns-specific PLC had no effect on intact platelets since the amount of SFLLR-induced P-selectin surface expression from intact platelets exposed to 5 units/ml PtdIns-specific PLC was 97 ± 3% of that from intact platelets not exposed to PtdIns-specific PLC. Similarly, PtdIns-specific PLC failed to inhibit Ca2+-induced P-selectin expression from platelets permeabilized with alpha -toxin. alpha -Toxin creates pores that restrict the entry of molecules greater than ~4.4 kDa (46). Ca2+-induced P-selectin expression from platelets permeabilized with alpha -toxin and subsequently exposed to 5 units/ml PtdIns-specific PLC was 101 ± 9% of that from alpha -toxin-permeabilized platelets not exposed to PtdIns-specific PLC. Thus, the effects of PtdIns-specific PLC on alpha -granule secretion require entry into the platelet cytosol. Inhibition of Ca2+-induced alpha -granule secretion by PtdIns-specific PLC occurred in a dose-dependent manner with an IC50 of ~0.5 units/ml (Fig. 3C). Inhibition of alpha -granule secretion by PtdIns-specific PLC was also time-dependent, with the amount of inhibition increasing to ~75% over a 45-min incubation time (Fig. 3D). These data demonstrate that incubation of SL-O-permeabilized platelets with PtdIns-specific PLC interferes with Ca2+-induced alpha -granule secretion.


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Fig. 3.   Effect of PtdIns-specific PLC on PtdIns(4,5)P2 synthesis in and Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets. A, 32P-labeled gel-filtered platelets (350 µl/sample) in PIPES/EGTA buffer were permeabilized with 3 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 15 min. Permeabilized platelets were then incubated in the presence of buffer (No Addition) or 5 units/ml PtdIns-specific PLC for 40 min. The lipids were extracted, and PtdIns(4,5)P2 levels were analyzed by TLC. Data are expressed as percent PtdIns(4,5)P2 levels compared with samples exposed to buffer alone. Error bars represent the S.E. of six samples. B, gel-filtered platelets were permeabilized with 3 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 15 min. Permeabilized platelets were then incubated for 15 min with buffer (No Addition), 5 units/ml PtdIns-specific PLC, or 5 units/ml heat-denatured PtdIns-specific PLC. Following this incubation, platelets were exposed to either buffer (No Ca2+) or 10 µM Ca2+ for 5 min. Platelets were then assayed for P-selectin expression by flow cytometry as described under "Experiment Procedures." Histograms represent the distribution of the relative fluorescence of 10,000 platelets. C, gel-filtered platelets were permeabilized with 3 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 15 min. Permeabilized platelets were then incubated with the indicated concentrations of PtdIns-specific PLC for 15 min. Platelets were exposed to 10 µM Ca2+ for 5 min and assayed for P-selectin expression. Data are expressed as the percent inhibition of P-selectin expression in samples exposed to PtdIns-specific PLC compared with samples exposed to buffer alone. Error bars represent the S.E. of three independent experiments. D, gel-filtered platelets were permeabilized with 3 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 15 min. Permeabilized platelets were then incubated with 5 units/ml PtdIns-specific PLC for the indicated times. After 1 h following permeabilization, platelets were exposed to 10 µM Ca2+ for 5 min and assayed for P-selectin expression. Data are expressed as the percent inhibition of P-selectin expression in samples exposed to PtdIns-specific PLC compared with samples exposed to buffer alone. Error bars represent the S.E. of three independent experiments.

Effect of Exogenous PtdIns(4,5)P2 on Ca2+-induced alpha -Granule Secretion-- The proposed role of PtdIns(4,5)P2 in granule secretion from neuroendocrine cells is to localize PtdIns(4,5)P2-binding proteins that mediate secretion to specific locations to permit membrane fusion (47). Consistent with this hypothesis, exogenously added PtdIns(4,5)P2 inhibits granule secretion from permeabilized PC12 cells (48). We therefore determined the effect of exogenously added PtdIns(4,5)P2 on platelet alpha -granule release. Platelets were permeabilized with SL-O for 15 min prior to the addition of either PtdIns(4,5)P2 or buffer. Following an additional 15-min incubation, platelets were exposed to buffer alone or Ca2+ for 5 min and subsequently analyzed for P-selectin surface expression by flow cytometry. Exogenously added PtdIns(4,5)P2 inhibited Ca2+-induced platelet alpha -granule secretion by ~80% (Fig. 4). PtdIns(4,5)P2 itself had little effect on alpha -granule secretion in the absence of exposure to Ca2+. The amount of SFLLR-induced P-selectin surface expression from intact platelets exposed to exogenously added PtdIns(4,5)P2 (50 µM) was 91 ± 2% of that from intact platelets not exposed to exogenously added PtdIns(4,5)P2. Similarly, Ca2+-induced P-selectin expression from platelets permeabilized with alpha -toxin and subsequently exposed to exogenously added PtdIns(4,5)P2 (50 µM) was 99 ± 6% of that from alpha -toxin-permeabilized platelets not exposed to exogenously added PtdIns(4,5)P2. Thus, the effects of exogenously added PtdIns(4,5)P2 require entry into the platelet cytosol. These data demonstrate that incubation of SL-O-permeabilized platelets with exogenous PtdIns(4,5)P2 inhibits Ca2+-induced alpha -granule secretion.


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Fig. 4.   Effect of exogenous PtdIns(4,5)P2 on Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets. A, gel-filtered platelets were permeabilized with 3 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 15 min. Permeabilized platelets were then incubated with buffer (No Addition) or 50 µM PtdIns(4,5)P2 for 15 min. Following this incubation, platelets were exposed to either buffer (No Ca2+) or 10 µM Ca2+ for 5 min. Platelets were then assayed for P-selectin expression by flow cytometry as described under "Experimental Procedures." Histograms represent the distribution of the relative fluorescence of 10,000 platelets. B, data are expressed as percent P-selectin surface expression compared with samples exposed to buffer alone. Error bars represent the S.E. of three independent experiments.

Effect of Wortmannin, LY294002, and Anti-PtdIns 3-Kinase Antibody on Ca2+-induced alpha -Granule Secretion-- PtdIns 3-kinase has previously been implicated in the process of regulated granule exocytosis from hematopoietic cells (49, 50). We therefore sought to determine whether PtdIns 3-kinase is involved in Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets. To this end, experiments using inhibitors of PtdIns 3-kinase, wortmannin and LY294002, were performed. In these experiments, neither 250 nM wortmannin nor 33.3 µM LY294002 inhibited Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets (Fig. 5A). The activity of 250 nM wortmannin and 33.3 µM LY294002 was confirmed in platelet aggregation studies. Reversible platelet aggregation, which is dependent on PtdIns 3-kinase activity (51), was inhibited to 36 ± 3% of base-line levels by 250 nM wortmannin and to 29 ± 6% of base-line levels by 33.3 µM LY294002. Thus, the concentrations of wortmannin and LY294002 used to assess the role of PtdIns 3-kinase in Ca2+-induced alpha -granule secretion are sufficient to inhibit PtdIns 3-kinase-dependent platelet functions. Antibodies directed at PtdIns 3-kinase p85alpha also failed to inhibit Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets (Fig. 5B). These results demonstrate that PtdIns 3-kinase is not involved in Ca2+-induced, ATP-dependent alpha -granule secretion.


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Fig. 5.   Effect of wortmannin, LY294002, and anti-PtdIns 3-kinase p85alpha antibody on Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets. A, gel-filtered platelets were incubated with 0.3% dimethyl sulfoxide (DMSO), 250 nM wortmannin, or 33.3 µM LY294002 for 30 min. Platelets were then permeabilized with 2 units/ml SL-O for 5 min and subsequently incubated with 5 mM MgATP at pH 6.9 for 25 min. Platelets were exposed to 10 µM Ca2+ for 5 min and analyzed for P-selectin expression by flow cytometry as described under "Experimental Procedures." Data are expressed as percent P-selectin surface expression compared with samples exposed to 0.3% dimethyl sulfoxide alone. B, gel-filtered platelets were incubated with MgATP in the presence of buffer (No addition), 40 µg/ml anti-PtdIns 3-kinase p85alpha monoclonal antibody, or 40 µg/ml nonimmune mouse IgG (nonimmune antibody). Platelets were subsequently permeabilized with 2 units/ml SL-O at pH 6.9 for 25 min. Platelets were then exposed to 10 µM Ca2+ for 5 min and analyzed for P-selectin expression by flow cytometry. Data are expressed as percent P-selectin surface expression compared with samples exposed to buffer alone.

Experiments performed in the presence of increasing concentrations of wortmannin demonstrated that wortmannin inhibited Ca2+-induced alpha -granule secretion to a maximum of ~50% at 3.125 µM (Fig. 6A). Similarly, 3.125 µM wortmannin inhibited the synthesis of PtdIns(4,5)P2 in SL-O-permeabilized platelets by ~50% (Fig. 6B). To determine whether exposure to wortmannin inhibits ATP- or Ca2+-dependent processes, SL-O-permeabilized platelets were exposed to wortmannin either 1) prior to ATP exposure or 2) after ATP exposure, but prior to Ca2+ exposure. Incubation of platelets with 3.125 µM wortmannin for 45 min inhibited Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets when wortmannin was added prior to ATP exposure (Fig. 6C). However, alpha -granule secretion was not inhibited in SL-O-permeabilized platelets exposed to ATP for 15 min and subsequently incubated with 3.125 µM wortmannin for 45 min (Fig. 6C). Thus, wortmannin inhibits ATP-dependent events, but not Ca2+-mediated triggering of alpha -granule secretion. The correlation between the reduction of PtdIns(4,5)P2 synthesis by micromolar concentrations of wortmannin and the inhibition of an ATP-dependent step of Ca2+-induced alpha -granule secretion is consistent with a role for PtdIns(4,5)P2 synthesis in this process.


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Fig. 6.   Effect of micromolar concentrations of wortmannin on Ca2+-induced alpha -granule secretion from and PtdIns(4,5)P2 synthesis in SL-O-permeabilized platelets. A, gel-filtered platelets were incubated with the indicated concentrations of wortmannin for 30 min. Platelets were then permeabilized with 2 units/ml SL-O for 5 min and subsequently incubated with 5 mM MgATP at pH 6.9 for 25 min. Platelets were exposed to 10 µM Ca2+ for 5 min and analyzed for P-selectin expression by flow cytometry as described under "Experimental Procedures." Data are expressed as percent P-selectin surface expression compared with samples exposed to dimethyl sulfoxide alone. Error bars represent the S.E. of three to seven independent experiments. B, 32P-labeled gel-filtered platelets (350 µl/sample) in PIPES/EGTA buffer were incubated with either 0.3% dimethyl sulfoxide (vehicle) or 3.125 µM wortmannin for 30 min. Labeled platelets were then permeabilized with 2 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 30 min. The lipids were extracted, and PtdIns(4,5)P2 levels were analyzed by TLC. Data are expressed as percent PtdIns(4,5)P2 levels compared with samples exposed to vehicle alone. Error bars represent the S.E. of six samples. C, one set of platelets was incubated with either vehicle (white bars) or 3.125 µM wortmannin (black bars) for 30 min. Platelets were subsequently permeabilized with 2 units/ml SL-O for 5 min and incubated with 5 mM MgATP at pH 6.9 for 15 min. A second set of platelets was permeabilized with 2 units/ml SL-O for 5 min and incubated with 5 mM MgATP at pH 6.9 for 15 min. Following incubation with MgATP, these samples were incubated with vehicle or 3.125 µM wortmannin for 45 min. Following incubation with MgATP, all samples were exposed to 10 µM Ca2+ for 5 min and analyzed for P-selectin surface expression. Data are expressed as percent P-selectin surface expression compared with samples exposed to vehicle. Error bars represent the S.E. of three independent experiments.

Effect of Anti-type II PIPK Antibodies on Ca2+-induced alpha -Granule Secretion-- PtdIns 5-phosphate levels increase in platelets following stimulation with thrombin (37). Furthermore, type II PIPK has previously been demonstrated to be abundant in platelets (38). We therefore determined the effect of anti-type II PIPK antibodies on PtdIns(4,5)P2 synthesis in and alpha -granule secretion from SL-O-permeabilized platelets. Anti-type II PIPK antibodies directed against the amino- and carboxyl-terminal ends of the alpha -isoform of human type II PIPK were used in these studies. The peptides used to generate these antibodies have no homology to type I PIPK. Each antibody recognized a single band of 53 kDa in platelet lysates (Fig. 7A). In contrast, nonimmune IgG failed to recognize any bands in platelet lysates (data not shown). In addition, both antibodies recognized a recombinant GST-tagged type II PIPK fusion protein of 79 kDa in immunoblot analysis (Fig. 7B). When incubated with MgATP-exposed platelets prior to permeabilization with SL-O, both anti-amino- and anti-carboxyl-terminal type II PIPK antibodies inhibited PtdIns(4,5)P2 levels by ~50% compared with permeabilized platelets incubated with nonimmune antibody (Fig. 7C). In contrast, neither anti-type II PIPK antibody inhibited PtdIns(4,5)P2 levels when incubated with platelets for 15 min following permeabilization with SL-O. We next determined the effect of anti-type II PIPK antibodies on Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets. In these experiments, platelets were incubated in the presence of nonimmune antibody or anti-type II PIPK antibody directed against either the amino- or carboxyl-terminal end of type II PIPK. Platelets were permeabilized with SL-O and then exposed to 10 µM Ca2+. Both antibodies significantly inhibited Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets (Fig. 7D). In contrast, nonimmune antibody had no effect on Ca2+-induced alpha -granule secretion. As observed with wortmannin, anti-type II PIPK antibodies inhibited Ca2+-induced alpha -granule secretion when added prior to, but not after, ATP (Fig. 7D). The amount of SFLLR-induced P-selectin surface expression from intact platelets exposed to anti-amino- or anti-carboxyl-terminal type II PIPK antibody was 104 ± 24 and 93 ± 18%, respectively, of that from intact platelets not exposed to anti-type II PIPK antibodies. Similarly, Ca2+-induced P-selectin expression from platelets permeabilized with alpha -toxin in the presence of anti-amino- or anti-carboxyl-terminal type II PIPK antibody was 100 ± 6 and 91 ± 22%, respectively, of that from alpha -toxin-permeabilized platelets not exposed to anti-type II PIPK antibodies. Thus, the effects of exogenously added anti-type II PIPK antibodies require entry into the platelet cytosol. We conclude that type II PIPK mediates alpha -granule secretion from Ca2+-induced SL-O-permeabilized platelets and acts prior to Ca2+ exposure.


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Fig. 7.   Effect of anti-type II PIPK antibodies on PtdIns(4,5)P2 synthesis in and Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets. A, proteins from human platelets were solubilized at 95 °C in sample buffer, separated by SDS-polyacrylamide gel electrophoresis on a 12% gel, and electrophoretically transferred to polyvinylidene difluoride membranes. Immunoblotting was performed with antibodies directed against the amino- and carboxyl-terminal peptides of type II PIPK as indicated. Bands were visualized using enhanced chemiluminescence for detection. The positions of the molecular mass standards used are indicated on the left. B, recombinant GST-tagged type II PIPK was solubilized at 95 °C in sample buffer, separated by SDS-polyacrylamide gel electrophoresis on a 12% gel, and electrophoretically transferred to polyvinylidene difluoride membranes. Immunoblotting was performed with antibodies directed against the amino- and carboxyl-terminal peptides as described for A. C, one set of 32P-labeled gel-filtered platelets (black bars) was permeabilized with 4 units/ml SL-O in the presence of 40 µg/ml nonimmune antibody, 40 µg/ml anti-amino-terminal type II PIPK antibody (N-term), or 40 µg/ml anti-carboxyl-terminal type II PIPK antibody (C-term) with 5 mM MgATP at pH 6.9 for 90 min. A second set of 32P-labeled platelets (white bars) was permeabilized with 4 units/ml SL-O in the presence of 5 mM MgATP alone at pH 6.9 for 15 min. This set of platelets was then exposed to 40 µg/ml nonimmune antibody, 40 µg/ml anti-amino-terminal type II PIPK antibody, or 40 µg/ml anti-carboxyl-terminal type II PIPK antibody for 75 min. The lipids were extracted, and PtdIns(4,5)P2 levels were analyzed by TLC. Data are expressed as percent PtdIns(4,5)P2 levels compared with samples exposed to nonimmune antibody. Error bars represent the S.E. of three to six samples. D, one set of gel-filtered platelets (black bars) was incubated in the presence of 40 µg/ml nonimmune antibody, 40 µg/ml anti-amino-terminal type II PIPK antibody, or 40 µg/µl anti-carboxyl-terminal type II PIPK antibody. Samples were then exposed to 10 mM MgATP and permeabilized with 5 units/ml SL-O. Following permeabilization, all samples were adjusted to pH 6.9 and subsequently exposed to 10 µM Ca2+. A second set of gel-filtered platelets (white bars) was incubated in the presence of 10 mM MgATP at pH 6.9 with no addition. Samples were then perme abilized with 5 units/ml SL-O. Following this MgATP exposure, samples from the second set were incubated with 40 µg/ml nonimmune antibody, 40 µg/ml anti-amino-terminal type II PIPK antibody, or 40 µg/µl of anti-carboxyl-terminal type II PIPK antibody, followed by 10 µM Ca2+. Following a 15-min incubation in the presence of Ca2+, all samples were assayed for P-selectin expression by flow cytometry as described under "Experimental Procedures." Data are expressed as percent P-selectin expression compared with samples incubated in the presence of buffer alone. Error bars represent the S.E. of four to eight independent experiments.

To assess the possibility that wortmannin and type II PIPK inhibit Ca2+-induced alpha -granule secretion by distinct mechanisms, we performed experiments using both inhibitors. In these experiments, we incubated platelets in the presence of 3.125 µM wortmannin or vehicle for 30 min and exposed them to nonimmune, anti-amino-terminal end, or anti-carboxyl-terminal end antibodies prior to ATP exposure and SL-O permeabilization. Under these conditions, ~40% inhibition of Ca2+-induced alpha -granule secretion was observed in platelets exposed to only wortmannin or either of the anti-type II PIPK antibodies compared with platelets treated with vehicle and nonimmune antibody (Fig. 8). Increasing the concentration of anti-type II PIPK antibodies to 200 µg/ml failed to increase the degree of inhibition of alpha -granule secretion. However, alpha -granule secretion from SL-O-permeabilized platelets exposed to both wortmannin and anti-type II PIPK antibodies was inhibited by ~70% compared with platelets exposed to vehicle and nonimmune antibody (Fig. 8). Thus, inhibition by wortmannin and anti-type II PIPK antibodies is additive.


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Fig. 8.   Effect of wortmannin and type II PIPK antibodies on Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets. Gel-filtered platelets were incubated in the presence (black bars) or absence (white bars) of 3.125 µM wortmannin for 30 min. Platelets were then exposed to 40 µg/ml nonimmune antibody or 40 or 200 µg/ml antibody directed against an amino-terminal (N-term) or carboxyl-terminal (C-term) peptide of type II PIPK as indicated and permeabilized with 4 units/ml SL-O in the presence of 5 mM MgATP. Following permeabilization, all samples were adjusted to pH 6.9. After a 30-min incubation, samples were exposed to 10 µM Ca2+ for an additional 10-min incubation. Samples were then analyzed for P-selectin expression by flow cytometry as described under "Experimental Procedures." Data are expressed as percent P-selectin expression compared with samples incubated in the presence of vehicle and nonimmune antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

One advantage of permeabilized secretory systems is that target-specific, cell-impermeable inhibitors such as antibodies and enzymes can be introduced intracellularly. This characteristic permits for more precise identification of molecules directly involved in the secretory process. The fact that Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets is inhibited by PtdIns-specific PLC (which cleaves PtdIns(4,5)P2), by exogenously added PtdIns(4,5)P2 (which may act by displacing proteins bound to endogenous PtdIns(4,5)P2), and by two different anti-type II PIPK antibodies (which inhibit PtdIns(4,5)P2 synthesis) provides strong evidence that PtdIns(4,5)P2 mediates alpha -granule secretion in this system. Since none of these inhibitors have inhibitory activity in intact platelets or in alpha -toxin-permeabilized platelets, access to the platelet cytosol is required for their inhibitory activity. The assertion that PtdIns(4,5)P2 mediates alpha -granule secretion is also supported by the observations that the ability to secrete alpha -granules upon Ca2+ exposure is temporally correlated with PtdIns(4,5)P2 synthesis and that micromolar concentrations of wortmannin inhibit both PtdIns(4,5)P2 synthesis and Ca2+-induced alpha -granule secretion.

A second advantage of permeabilized secretory systems is that they allow for the dissection of the secretory process into component parts. Like other permeabilized cells, platelets will undergo Ca2+-mediated secretion only following exposure to ATP (42, 43, 46). SL-O-permeabilized platelet systems differ from many permeabilized cell systems, however, in that Ca2+-induced granule secretion from platelet systems does not exhibit a requirement for exogenous cytosolic proteins (12, 45, 52). The observation that PtdIns(4,5)P2 synthesis occurs in SL-O-permeabilized platelets in the absence of exogenous cytosol suggests that some PtdIns(4,5)P2 synthetic machinery remains cell-associated in this permeabilized secretory system. In our studies, inhibitors of PtdIns(4,5)P2 synthesis inhibited alpha -granule secretion when added prior to ATP exposure, but not when added following ATP exposure, but prior to Ca2+-mediated triggering of secretion. This finding raises the possibility that activation-dependent changes in localized PtdIns(4,5)P2 concentrations mediate granule secretion. Indeed, activation of platelets is accompanied by both an increase in PtdIns kinase activity (33, 35, 36) and relocalization of PtdIns kinases (53). Future studies will determine whether activation-dependent increases in PtdIns(4,5)P2 at zones of granule secretion within the platelet are necessary for alpha -granule secretion.

Previous studies of hematopoietic cells have implicated PtdIns 3-kinase in the process of regulated granule exocytosis. For example, nanomolar concentrations of wortmannin inhibit granule secretion from a basophilic leukemia cell line (RBL-2H3) (49) and from natural killer cells (50). However, the fact that nanomolar concentrations of wortmannin, 33.3 µM LY294002, and antibodies directed at PtdIns 3-kinase p85alpha did not inhibit secretion from SL-O-permeabilized platelets (Fig. 5) demonstrates that D3 phosphoinositides are not involved in alpha -granule secretion from platelets. The observation that wortmannin failed to inhibit alpha -granule secretion at concentrations that inhibit PtdIns 3-kinase is consistent with the data of Kovacsovics et al. (51). Their studies using intact platelets demonstrated that although wortmannin at nanomolar concentrations inhibits thrombin receptor agonist peptide-induced activation of glycoprotein IIb-IIIa, it fails to affect alpha -granule secretion from platelets. Similarly, wortmannin at nanomolar concentrations is a relatively poor inhibitor of neutrophil granule secretion, even though it inhibits the neutrophil oxidative burst with an IC50 of <5 nM (54). Thus, granule secretion from various cells of hematopoietic origin differs in requirements for phosphoinositides, and platelet alpha -granule secretion demonstrates a requirement for D4, but not D3, phosphoinositides.

In addition to demonstrating that PtdIns(4,5)P2 is synthesized in SL-O-permeabilized platelets, these studies demonstrate that the type II PIPK-dependent pathway is involved in the synthesis of PtdIns(4,5)P2 required for Ca2+-induced alpha -granule secretion. In PC12 cells, type I, but not type II, PIPK reconstitutes Ca2+-mediated secretion (30). To our knowledge, type II PIPK has not previously been demonstrated to function in a cellular process (55). However, PtdIns 5-kinase levels are increased upon exposure of intact platelets to thrombin (37). In this study, we show that anti-type II PIPK antibodies partially inhibit Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets. Three isoforms of type II PIPK (alpha , beta , and gamma ) have been identified. The antibodies used in this study are directed to the amino- and carboxyl-terminal ends of the alpha -isoform. However, these antibodies cross-react with the beta -isoform (data not shown); and thus, we are not able to determine which isoform(s) of type II PIPK is active in platelets. Anti-type II PIPK antibodies do not cause complete inhibition of Ca2+-induced alpha -granule secretion from SL-O-permeabilized platelets. The degree of inhibition of secretion was no greater when the concentration of antibody was increased from 40 to 200 µg/ml (Fig. 8). Neither the anti-amino- nor anti-carboxyl-terminal type II PIPK antibody is directed against the proposed active site of the kinase. Thus, it is possible that the antibodies cause partial inhibition of type II PIPK function by disrupting its interactions with other molecules (e.g. PtdIns kinases) required for PtdIns(4,5)P2 production (56). Another possibility is that partial PtdIns(4,5)P2 production in the presence of anti-type II PIPK antibodies is secondary to the activity of type I PIPK isoforms, which are also found in platelets.2

The fact that exposure of SL-O-permeabilized platelets to micromolar concentrations of wortmannin results in a 50% reduction of both PtdIns(4,5)P2 synthesis and Ca2+-induced, ATP-dependent alpha -granule secretion when added prior to, but not after, ATP exposure raises the possibility that the PtdIns 4-kinase pathway is also involved in alpha -granule secretion. Three isoforms of PtdIns 4-kinase have been identified and can be distinguished based on their sensitivity to wortmannin. A 55-kDa PtdIns 4-kinase is resistant to wortmannin (57). A 230-kDa PtdIns 4-kinase alpha  with a 97-kDa splice variant is inhibited by wortmannin with an IC50 of ~1 µM and is nearly completely inhibited at 10 µM (58). PtdIns 4-kinase beta  is inhibited by wortmannin with an IC50 of ~120 nM and is completely inhibited at 1 µM. However, wortmannin at micromolar concentrations is not specific for PtdIns 4-kinases. Inhibition of the ATP-dependent step of Ca2+-induced alpha -granule secretion by micromolar concentrations of wortmannin may occur by interfering with molecules not involved in PtdIns(4,5)P2 synthesis. The fact that PtdIns(4,5)P2 synthesis is also inhibited by wortmannin may be secondary to inhibition of a PtdIns 5-kinase. Although the PtdIns 5-kinases in platelets have not been well characterized, a 235-kDa mammalian PtdIns kinase originally discovered in insulin-sensitive cells (59) was subsequently found to synthesize PtdIns 5-phosphate (60). Wortmannin inhibits this PtdIns 5-kinase with an IC50 of ~600 nM and completely inhibits kinase activity at 4 µM wortmannin (60). Thus, it is possible that the effect of wortmannin on PtdIns(4,5)P2 synthesis and Ca2+-induced alpha -granule secretion is via inhibition of a PtdIns 5-kinase pathway rather than through a PtdIns 4-kinase pathway. However, the identification of the complete set of PtdIns kinase and PIPK isoforms used in the synthetic pathway of PtdIns(4,5)P2 required for alpha -granule secretion remains to be determined.

How might PtdIns(4,5)P2 mediate alpha -granule secretion? Platelet alpha -granule secretion is known to require the SNARE protein machinery (12-15). In other secretory systems, it has been postulated that PtdIns(4,5)P2 might act by interacting with proteins, such as synaptotagmin and Munc-18-interacting protein, that both bind PtdIns(4,5)P2 and interact with SNARE proteins (61). In addition, PtdIns(4,5)P2 interacts with ADP-ribosylation factors and GTPase-activating proteins to localize these molecules to sites of membrane fusion (61). However, the presence of ADP-ribosylation factors in platelets and the role of GTPase-activating proteins and ADP-ribosylation factors in platelet secretion remain to be explored. PtdIns(4,5)P2 also stimulates phospholipase D (62), which generates phosphatidic acid and is thought to influence platelet granule secretion (24). Phosphatidic acid, in turn, stimulates type I PIPK activity (63). Thus, a feed-forward mechanism is established whereby cell activation leads to increased synthesis of phosphatidic acid and PtdIns(4,5)P2. Future studies will provide a more detailed understanding of how PtdIns(4,5)P2 mediates interactions between platelet signaling events and SNARE protein rearrangements that result in membrane fusion.

    ACKNOWLEDGEMENTS

We thank Christopher Carpenter for helpful discussion, advice, and critical reading of the manuscript and Bruce Furie for critical reading of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Grant HL63250, Burroughs Wellcome Fund Career Awardee, and participant in the Beth Israel Deaconess Medical Center-Harvard/Massachusetts Institute of Technology Health Sciences and Technology Clinical Investigator Training Program in collaboration with Pfizer. To whom correspondence should be addressed: RE 319, Research East, P. O. Box 15732, Boston, MA 02215. Tel: 617-667-0627; Fax: 617-975-5505; E-mail: rflaumen@caregroup.harvard.edu.

Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M008184200

2 C. L. Carpenter, personal communication.

    ABBREVIATIONS

The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor; PLC, phospholipase C; PtdIns, phosphatidylinositol; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PIPK, phosphatidylinositol-phosphate kinase; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); SL-O, streptolysin O; GST, glutathione S-transferase; PIPES, 1,4-piperazinediethanesulfonic acid.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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