Nitric Oxide Inhibits Thrombin Receptor-activating Peptide-induced Phosphoinositide 3-Kinase Activity in Human Platelets*

Although nitric oxide (NO) has potent antiplatelet actions, the signaling pathways affected by NO in the platelet are poorly understood. Since NO can induce platelet disaggregation and phosphoinositide 3-kinase (PI3-kinase) activation renders aggregation irreversible, we tested the hypothesis that NO exerts its antiplatelet effects at least in part by inhibiting PI3-kinase. The results demonstrate that the NO donorS-nitrosoglutathione (S-NO-glutathione) inhibits the stimulation of PI3-kinase associated with tyrosine-phosphorylated proteins and of p85/PI3-kinase associated with the SRC family kinase member LYN following the exposure of platelets to thrombin receptor-activating peptide. The activation of LYN-associated PI3-kinase was unrelated to changes in the amount of PI3-kinase physically associated with LYN signaling complexes but did require the activation of LYN and other tyrosine kinases. The cyclic GMP-dependent kinase activator 8-bromo-cyclic GMP had similar effects on PI3-kinase activity, consistent with a model in which the cyclic nucleotide mediates the effects of NO. Additional studies showed that wortmannin and S-NO-glutathione have additive inhibitory effects on thrombin receptor-activating peptide-induced platelet aggregation and the surface expression of platelet activation markers. These data provide evidence of a distinct and novel mechanism for the inhibitory effects of NO on platelet function.

By inhibiting platelet function, nitric oxide (NO) 1 is believed to play a role in regulating thrombosis and hemostasis. However, the signaling pathways underlying the inhibitory action of NO on the platelet are poorly understood. One signaling event that appears to be critical to thrombin-induced platelet activation is the stimulation of phosphoinositide 3-kinase (PI3-kinase). Thus, the fungal metabolite wortmannin, an irreversible inhibitor of PI3-kinase, mimics some of the physiological and biochemical effects of NO on the platelet, such as inhibition of thrombin receptor-activating peptide (TRAP)-induced platelet aggregation and the surface expression of activated integrin glycoprotein (GP) IIb-IIIa, the principal fibrinogen receptor (1).
PI3-kinase represents a family of ubiquitous enzymes involved in the regulation of mitogenesis, vesicular trafficking, glucose transport, cytoskeletal rearrangements, and other cellular functions. PI3-kinase catalyzes the phosphorylation of the inositol ring at the D3 position in a variety of phosphoinositide substrates forming 3-phosphorylated phosphoinositides (2,3); some isoforms can also catalyze the serine phosphorylation of themselves and other proteins (4,5).
The p85/PI3-kinase, the first to be described in the platelet, is a heterodimer composed of a p85 regulatory subunit containing two SRC homology (SH) 2 domains and one SH3 domain, and a catalytic p110 subunit. Its "classical" mechanism of activation, originally described as the mechanism by which it is stimulated in lymphocytes by large T antigen and activation of receptor tyrosine kinases, involves the interaction of one or both SH2 domains with phosphotyrosine moieties on other proteins (6 -8). The p85/PI3-kinase is also activated by a number of G protein-linked receptors, including the thrombin receptor in platelets (9,10). This type of activation may involve either a non-receptor tyrosine-phosphorylated intermediate (11) or the recently described direct activation of some isoforms by G protein ␤␥ subunits (12,13). A second class of PI3-kinase, PI3-kinase-␥, has also been recently identified in platelets. It appears to consist of unique p110 catalytic and p101 regulatory subunits (14 -17) and is activated only by G protein ␤␥ subunits.
Exposure of platelets to thrombin or the thrombin receptoractivating peptide (TRAP) results in the activation and translocation of one or both types of PI3-kinase to the cytoskeleton at sites of integrin-dependent focal adhesions, where the enzyme is thought to have an important function in the cytoskeletal reorganization that accompanies irreversible platelet aggregation and clot retraction (1, 18 -19). More recent studies indicate that the p85/PI3-kinase, rather than the PI3-kinase-␥, is involved in the activation of the integrin GPIIb-IIIa in platelets (17). We have previously shown that nitric oxide donors can inhibit GPIIb-IIIa activation and fibrinogen binding (20,21). Accordingly, we investigated the effects of NO on components of the p85/PI3-kinase pathway in an effort to identify a new mechanism by which NO can inhibit platelet function. fluoride, benzamidine, 8-Br-cyclic GMP, glutathione, and all other common reagents were obtained from Sigma. Sodium orthovanadate was purchased from Aldrich. TRAP was obtained from Bachem (King of Prussia, PA). Protein A-Sepharose was purchased from Amersham Pharmacia Biotech. [␥-32 P]ATP was obtained from NEN Life Science Products. Liver phosphatidylinositol was purchased from Avanti Biotechnology (Alabaster, AL). Enhanced chemiluminescence peroxidase detection kits were obtained from Amersham Pharmacia Biotech or Pierce.
Platelet Preparation-Using standard phlebotomy techniques, human blood was obtained from healthy volunteers who had not taken any antiplatelet medication for at least 10 days. The blood was centrifuged (150 ϫ g, 11 min, 22°C), and the upper one-half to two-thirds of the platelet-rich plasma supernatant was collected for experimental use. The remaining blood was further centrifuged at 3000 ϫ g to collect platelet-poor plasma in the supernatant. Platelet-poor plasma was occasionally used to dilute samples in order to equalize the number of platelets per tube. When handling blood, extreme care was taken to prevent shear-induced platelet activation. To prepare gel-filtered platelets, platelet-rich plasma was passed over a Sepharose 2B column, as described previously (22). Platelet counts were determined using a Coulter counter (Coulter Electronics, Miami, FL).
Immunoprecipitation and Western Blotting-Gel-filtered platelets or platelet-rich plasma were kept at room temperature for 30 min prior to any experimental manipulation; subsequently, platelets were counted, and the number of cells in each tube was adjusted with platelet-poor plasma or buffer as necessary. Platelets were stimulated under stirring conditions in an aggregometer for the indicated times. Aggregation was stopped by adding 2ϫ ice-cold lysis buffer containing 2% Triton X-100, 20% glycerol, 50 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM EDTA, 10 mM sodium pyrophosphate, 100 mM NaF, 0.1 mg/ml aprotinin, 1 mg/ml leupeptin, 0.35 mg/ml phenylmethylsulfonyl fluoride, 10 mM benzamidine, and 2 mM sodium orthovanadate. The tubes were kept on ice for 15 min and mixed in a rotor for 10 min at 4°C; the Triton-soluble fraction was collected by centrifuging the lysate at 15,400 ϫ g for 4 min. The supernatant was then subjected to immunoprecipitation by adding a primary antibody at a titer specified by the manufacturer and 100 l of a 1:1 PBS/protein A-Sepharose slurry, after which the mixture was incubated overnight at 4°C. The next day, the beads were washed three times with a buffer containing 1% Nonidet P-40 in PBS, three times with 0.5 M LiCl in 0.1 M Tris, pH 7.5, and two times with 100 mM NaCl, 1 mM EDTA in 10 mM Tris.
After the last wash, 50 l of 1ϫ SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer was added to the beads, and the tubes were boiled for 3 min in a water bath. Proteins were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin in PBS/ Tween overnight and probed with primary antibodies for 6 h. As secondary antiserum of horseradish peroxidase-coupled anti-rabbit antibody (Amersham Pharmacia Biotech) was next used for identifying bands; the immunoreactive products were visualized with the ECL chemiluminescence system (Amersham Pharmacia Biotech) and analyzed with a densitometer from PDI Imageware Systems (Huntington Station, NY).
Protein Kinase Assay-Protein kinase assays were carried out on LYN immunoprecipitates. After the last wash, kinase reactions were initiated by adding 20 l of 25 mM HEPES, 100 mM NaCl, 1 mM sodium orthovanadate, 3 mM MnCl 2 , 10 mM MgCl 2 , 0.5 M ATP, and 20 Ci of [␥-32 P]ATP (6,000 mCi/mmol) to the bead-bound immunoprecipitates. The reactions were stopped after 10 min by the addition of 1 ml of ice-cold PBS/5 mM EDTA, and the beads were washed once with the same solution. After the wash, SDS-PAGE loading buffer was added to the beads, and the reaction tubes were boiled for 3 min. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes as for Western blotting, and the labeled kinase substrates were visualized by autoradiography.
For LYN kinase assays using histone H1 as a substrate in LYN immunoprecipitates, 30 l of the foregoing kinase reaction mixture containing 50 g/l histone type III-SS (Sigma) were added. (The histone type III-SS served as a source of histone H1.) The reaction was stopped after 10 min by adding 1 ⁄3 volume of 3ϫ loading buffer, and the phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography.
Phosphatidylinositol 3-Kinase Assay-The PI3-kinase activity in immunoprecipitates was determined as described previously (23). Briefly, the Sepharose beads were washed as specified for the protein kinase assay. The substrate for PI3-kinase, liver phosphatidylinositol (PI), was prepared as follows. Twenty micrograms per sample of PI in chloroform were dried under a gentle stream of argon. The lipids were then resuspended in 10 l of 10 mM Tris, pH 7.5, and 1 mM EGTA. This PI mix was added to each tube together with 50 l of the third washing buffer described above and 10 l of 100 mM MgCl 2 . The reaction was then initiated with the addition of 5 l of a solution composed of 20 mM MgCl 2 , 0.88 mM ATP, and 2 Ci of [␥-32 P]ATP. Incubations were carried out for 10 min and stopped with the addition 20 l of 8 M HCl and 160 l of chloroform:methanol (1:1). The tubes were then centrifuged to separate the aqueous and organic phases, and the lipid products were resolved by thin layer chromatography with a running solvent composed of 120 ml of chloroform, 94 ml of methanol, 23 ml of water, and 4 ml of ammonium hydroxide. The radioactive PI3-P product was visualized by autoradiography and quantitated by Cerenkov counting.
Flow Cytometry-Two-color flow cytometric analysis was used to determine the activation of platelet surface glycoprotein IIb-IIIa (the fibrinogen receptor integrin II b ␤ 3 ) or the platelet surface expression of P-selectin. Platelet-rich plasma was incubated with buffer or 100 nM wortmannin for 30 min or 10 M S-NO-glutathione for 10 min or both S-NO-glutathione and wortmannin. Subsequently, 15 l of platelet-rich plasma diluted 1:20 in modified HEPES-Tyrode's buffer, pH 7.4 (HT), were incubated, in the presence of appropriate antibody combinations, with 2.5 mM glycyl-L-prolyl-L-arginyl-L-proline (Calbiochem) to prevent fibrin polymerization, and either thrombin or TRAP at the concentrations indicated in the figure legends. FITC-conjugated monoclonal antibody PAC1 was used to measure activation of the GPIIb-IIIa complex, with the CD41-specific monoclonal antibody P2 conjugated to phycoerythrin used as the specific platelet label. Platelet surface P-selectin exposure was measured by biotin-conjugated monoclonal antibody S12, with the CD61-specific monoclonal antibody Y2/51-FITC used as the specific platelet label. Incubations were carried out for 10 min. After adding 30 g/ml phycoerythrin-streptavidin (Jackson ImmunoResearch, West Grove, PA) to samples containing S12-biotin, samples were fixed with 1% formaldehyde for 20 min at room temperature and diluted with 9 volumes of HT. PAC1-FITC samples, in contrast, were fixed directly and diluted with 9 volumes of HT.
Flow cytometry was performed in an EPICS Profile II flow cytometer (Coulter, Miami, FL). FITC and phycoerythrin fluorescence was determined with 525-and 575-nm band pass filters, respectively. Platelets were identified by gating on their characteristic light scatter and positive FITC fluorescence for P-selectin analysis and phycoerythrin fluorescence for GPIIb-IIIa analysis, as described previously (25); subsequently, platelets were analyzed for binding of either the biotinylated S12 or FITC-conjugated PAC1 antibodies. P-selectin is expressed only after platelet degranulation, and PAC1 recognizes only the fibrinogenbinding site in activated GPIIb-IIIa complexes and, therefore, in contrast to P2 and Y2/51, does not bind to resting platelets. Background binding values were obtained by analyzing platelets stained with appropriate nonspecific isotype controls.
Statistical Analysis-Data are expressed as mean Ϯ S.E. Data were one-or two-way analysis of variance as appropriate. The results of an analysis were considered significant when p values were Յ0.05.

Inhibition of TRAP-stimulated p85/PI3-Kinase Activity in
Platelets by NO-The p85/PI3-kinase is stimulated primarily when its SH2 domains bind phosphotyrosine moieties on other proteins (6 -8). To determine if NO has an effect on PI3-kinase activation, we therefore examined the effect of the NO donor S-nitrosoglutathione (S-NO-glutathione, 10 M) on antiphosphotyrosine-immunoprecipitable PI3-kinase activity. Control platelets and platelets pretreated with S-NO-glutathione were incubated for 1 min with TRAP, and lipid kinase assays were carried out on antiphosphotyrosine immunoprecipitates from Triton X-100-soluble extracts. Although PI3-kinase has been reported to translocate into the cytoskeletal fraction after TRAP stimulation, this process required longer than 1 min (19), an observation that was confirmed in control studies (data not shown). As shown in Fig. 1A (left panel), TRAP significantly stimulated phosphotyrosine-associated PI3-kinase activity in the soluble fraction compared with that in unactivated platelets (CON), and this effect was blocked by preincubation of platelets with S-NO-glutathione (TRAP ϩ S-NO).
To identify specific tyrosine kinases involved in the thrombin-induced activation of platelet p85/PI3-kinase, we carried out lipid kinase assays on immunoprecipitates of the SRC family kinase LYN. Following platelet activation, LYN is activated and associates with several signaling molecules that are known to be present in complexes with activated PI3-kinase, including focal adhesion kinase (26), the SRC family kinase SYK (11,26), and several membrane glycoproteins involved in outside-in signaling, including GPIIb-IIIa (27)(28)(29). Moreover, the binding of the SH3 domain of LYN to purified PI3-kinase leads to a severalfold increase in its specific activity (30). Fig.  1A (right panel) shows a 4-fold increase in LYN-associated PI3-kinase activity after TRAP stimulation compared with unactivated platelets (CON), which was significantly attenuated by preincubation with S-NO-glutathione (TRAP ϩ S-NO). Similar inhibitory effects were noted using a different NO donor, diethylamino-NONOate (data not shown). Thus, at least one PI3-kinase target subpopulation for the inhibitory effect of NO is the LYN-associated PI3-kinase pool.
To determine if the effect of NO on phosphotyrosine and LYN-associated PI3-kinase was due to nonspecific inhibition of total PI3-kinase, total PI3-kinase activity was assayed in p85 regulatory subunit immunoprecipitates from TRAP-stimulated platelets that had been previously incubated with or without S-NO-glutathione. In contrast to the results in phosphotyrosine and LYN immunoprecipitates, total PI3-kinase activity decreased by 38% upon exposure to TRAP and, more importantly, S-NO-glutathione had no effect on p85 activity, either by itself (S-NO) or in combination with TRAP (TRAP ϩ S-NO) (Fig. 1B). Since the activity of the PI3-kinase that coimmunoprecipitated with LYN was between 0.7 and 3.1% of the p85-immunoprecipitable activity, and since both the p85 and LYN immunoprecipitations were essentially quantitative (see Fig. 3B), the results in Fig. 1 are consistent with the hypothesis that TRAP selectively activates and NO inhibits only a small subpopulation of the p85/PI3-kinase enzyme, including that in LYN signaling complexes. Although it is not known what other signaling complexes that contain PI3-kinase are affected by S-NOglutathione, it should be noted that SRC itself, as a prototypical SRC kinase family member, did not associate with measurable PI3-kinase activity in immunoprecipitates under any condition (data not shown).
Association of p85 and LYN Is Not Altered by NO- Fig. 2 shows an anti-p85 Western blot that demonstrates the presence of p85 in LYN immunoprecipitates from resting platelets (CON) and platelets preincubated with buffer (TRAP) or S-NOglutathione (TRAP ϩ S-NO) for 10 min and then stimulated with TRAP. Densitometry showed that the amount of PI3kinase associated with LYN remains constant regardless of agonist exposure or preincubation with S-NO-glutathione, indicating that neither thrombin nor NO affects the physical association of the two enzymes. Similar results were obtained in Western analyses of LYN-associated PI3-kinase in neutrophils. 2 The data in Fig. 1 suggest that LYN-associated PI3-kinase accounts for only a fraction of the total PI3-kinase pool. To confirm this hypothesis, LYN and p85 Western blots were carried out on platelet extracts before and after LYN was removed by immunoprecipitation. The result (Fig. 3A) indi-2 G. M. Bokoch, personal communication.
FIG. 1. PI3-kinase activity in phosphotyrosine, LYN, and p85 immunoprecipitates. A, gel-filtered platelets (500 l) were stimulated with 10 M TRAP for 1 min after a 10-min preincubation with 10 M S-NO-glutathione (S-NO) or buffer. Unstimulated platelets preincubated with buffer served as controls (CON). Aggregations were followed in an aggregometer in one tube (extent of aggregation ranged from 50 to 80%), and the reaction was stopped in the others by the addition of an equal volume of ice-cold homogenization buffer. After platelet lysis, immunoprecipitations were carried out with either antiphosphotyrosine (left panel, PY) or anti-LYN (right panel, LYN) antibodies as described under "Experimental Procedures." PI3-kinase assays were performed on the immunoprecipitates as described under "Experimental Procedures." The values in each experiment were subsequently normalized to those in the control condition. The results are presented as the average of four separate experiments (ϮS.E.), each performed in duplicate. B, gel-filtered platelets were preincubated for 10 min with S-NO-glutathione or buffer and then stimulated with TRAP for 1 min. PI3-kinase activity in anti-p85 immunoprecipitates was measured and presented as in phosphotyrosine and LYN immunoprecipitates. In absolute terms, the activity in LYN immunoprecipitates averaged between 0.7% (CON) and 3.1% (TRAP) that in p85 immunoprecipitates. The results are presented as the average of five experiments (ϮS.E.). *, p Ͻ 0.05 compared with control platelets; **, p Ͻ 0.05 compared with TRAP-stimulated platelets; each by repeated measures ANOVA. cated that, despite the complete loss of all of the upper band and most of the LYN lower band to the immunoprecipitate, the quantity of p85 in the extract did not perceptibly change. Thus, the pool of p85/PI3-kinase associated with LYN is a very small percentage of the total. Similar immunoabsorption studies were conducted to assess the relative size of the pool of LYN that was associated with PI3-kinase. LYN Western blots were conducted on platelet extract proteins before and after p85 was removed by immunoprecipitation. The results, shown in Fig.  3B, indicate that all of the LYN upper band and most of the LYN lower band were lost during the p85 immunoprecipitation, i.e. became p85-associated. These results indicate that PI3-kinase is an integral component of cytoplasmic LYN signaling complexes and that little LYN is present in complexes that do not contain PI3-kinase.
LYN can associate with PI3-kinase either directly via an interaction between the p85 proline-rich domain and LYN SH3 domain (30) or indirectly in a protein complex in which the two are bridged by c-CBL (31). In order to determine if the latter type of interaction occurs in platelets, c-CBL Western blots were performed on LYN immunoprecipitates obtained from TRAP-stimulated and unstimulated platelets. Fig. 4 demonstrates that c-CBL was detectable in cell extracts as well as in LYN immunoprecipitates. Thus, in at least some of the signal-ing complexes containing PI3-kinase and LYN, the two are likely to be bridged by c-CBL.
Effect of Nitric Oxide on LYN-associated PI3-Kinase Involves cGMP-dependent Protein Kinase-In order to test the hypothesis that the effect of NO on LYN-associated PI3-kinase is mediated by cGMP-dependent protein kinase, platelets were preincubated with the cGMP analog 8-bromo-cyclic GMP (8-Br-cGMP) for 30 min, and PI3-kinase activity was measured in LYN immunoprecipitates. Fig. 5 shows that 8-Br-cGMP inhibited the stimulation by TRAP of LYN-associated PI3-kinase activity to a similar extent as S-NO-glutathione, indicating that the NO effect was mediated, at least in part, by cGMP-dependent protein kinase.
Modulation of LYN Kinase Activity by TRAP and NO-The most direct mechanism by which TRAP may stimulate LYNassociated PI3-kinase activity is that LYN becomes activated by upstream signaling elements and then itself activates PI3kinase. To confirm that LYN, indeed, becomes activated, we assessed LYN kinase activity using an in vitro histone H1 phosphorylation assay. As shown in Fig. 6A, the phosphorylation of histone H1 increased 2-fold in TRAP-stimulated platelets (TRAP) as compared with unstimulated platelets (CON), an effect that was blocked by S-NO-glutathione (TRAP ϩ S-NO). Thus, LYN is activated by TRAP, and this activation is blocked by S-NO-glutathione.
In the process of obtaining LYN kinase activity measurements, we observed that a number of proteins in the LYN immunoprecipitates were phosphorylated in vitro. Fig. 6B is an autoradiogram showing the results of in vitro protein kinase assays in LYN immunoprecipitates from resting platelets (CON) and platelets stimulated with TRAP after a 10-min preincubation with buffer (TRAP) or S-NO-glutathione (TRAP ϩ S-NO). Interestingly, p53/p56 LYN was less phosphorylated in TRAP-stimulated than in control platelets, whereas S-NOglutathione partially blocked the effect of TRAP. These effects were not due to changes in the quantity of LYN protein in the immunoprecipitate, as shown in Fig. 6C. In contrast, TRAP stimulation increased the phosphorylation of a 70-kDa protein running just above LYN that was completely blocked by S-NOglutathione preincubation. The identity of this protein remains to be determined; Western blots have indicated that it is neither the SRC family kinase SYK, which is known to be associated with LYN, nor paxillin, a major docking protein in the cytoskeletal network (data not shown). Fig. 6D demonstrates the lack of kinase activity in control immunoprecipitates using anti-GLUT4 antibody (IF8) or only protein A-Sepharose (Prot A). Taken together, these data demonstrate that LYN signaling complexes either contain one or more TRAP-activated phosphatases or contain other kinases that phosphorylate LYN and PI3-kinase and are inhibited by TRAP.
To determine if activation of LYN and other tyrosine kinases is required for the activation of PI3-kinase in LYN signaling complexes, platelets were preincubated for 30 min with the tyrosine kinase inhibitor genistein (5 M) or the SRC family kinase-selective inhibitor PP2, and PI3-kinase activity was measured in LYN immunoprecipitates. As shown in Fig. 7, TRAP stimulated LYN-associated PI3-kinase 2-3-fold. Genis-tein had a small stimulatory effect (60%) on LYN-associated PI3-kinase when added alone but completely blocked its activation by TRAP (Fig. 7A). Similarly, Fig. 7B demonstrates that PP2 blunted the stimulation of TRAP of PI3-kinase, while having no significant effect of its own. The data indicate that activation of an SRC family kinase, most likely LYN, is necessary for the stimulation of LYN-associated PI3-kinase.
Effects of NO and Wortmannin on Platelet Aggregation and Surface Glycoprotein Expression-Since cGMP-dependent protein kinase activation is known to inactivate the protein kinase C (PKC)/inositol trisphosphate pathway, PI3-kinase is not likely to be the only site at which NO donors act to inhibit platelet activation (20). For this reason, we evaluated the antiplatelet effects of wortmannin, a relatively selective PI3-kinase inhibitor (at concentrations Ͻ100 nM), and S-NO-glutathione, used alone and in combination. We reasoned that if the S-NO-glutathione inhibited platelet aggregation principally through its effect on PI3-kinase, then the two treatments would not be additive. The inhibitory dose responses for S-NO-glutathione and wortmannin were first determined (wortmannin, 0.1 nM to 1 M; S-NO-glutathione, 0.1 nM to 10 M). Fig. 8 shows that wortmannin inhibited TRAP-induced aggregation by over 80% at 100 nM and that its IC 50 was 10 nM. For S-NO-glutathione, there was nearly complete inhibition of aggregation by concentrations as low as 1 M, and its IC 50 was 120 nM. We found an additive response when platelets were preincubated in the presence of 10 nM wortmannin and 10 nM S-NO-glutathione or with 10 nM wortmannin and 100 nM S-NO-glutathi- Gel-filtered platelets were preincubated with vehicle or 1.5 mM 8-Br-cGMP for 30 min prior to stimulation with TRAP as in Fig. 1. Resting gel-filtered platelets served as controls (CON). Duplicate determinations of PI3-kinase activity in LYN immunoprecipitates were performed as in Fig. 1. The figure shows the average results from three experiments (ϮS.E.). *, p Ͻ 0.05 compared with control platelets; **, p Ͻ 0.05 compared with TRAP-stimulated platelets; each by repeated measures ANOVA.
FIG. 6. Phosphorylation of exogenous and endogenous proteins in LYN immunoprecipitates. A, protein kinase assays were conducted with anti-LYN immunoprecipitates from resting platelets (CON), platelets stimulated with 10 M TRAP for 1 min (TRAP), or platelets preincubated with 10 M S-NO-glutathione for 10 min prior to TRAP stimulation (TRAP ϩ S-NO). Histone III-SS was added as a source of histone H1, the exogenous substrate. The reaction products were separated by SDS-PAGE and transferred to nitrocellulose, and the radiolabeled kinase products were visualized by autoradiography. The figure shows an autoradiogram from a typical experiment (n ϭ 3). B, assays were carried out in immunoprecipitates in the absence of histone to identify endogenous LYN-associated substrates for kinases in the immunoprecipitate. A typical autoradiogram is presented (n ϭ 4). C, the nitrocellulose membrane in Fig. 5B was blotted with anti-LYN antibody, and LYN was visualized. D, specificity of the immunoprecipitate. A LYN immunoprecipitate (LYN) kinase assay is shown in the right lane. The left lane shows a similar assay carried out in an immunoprecipitate made with anti-GLUT4 (IF8), whereas the center lane shows an assay carried out when only the protein A-Sepharose beads (Prot A) were exposed to extract.  TRAP ϩ GEN, respectively). B, similar experiments were carried out using the SRC family kinase inhibitor PP2 (5 M). Preincubation with PP2 was also performed for 30 min. PI3-kinase activity was then measured in LYN immunoprecipitates as in Fig. 1. Results are presented as the average of three experiments for genistein and four experiments for PP2 (ϮS.E.). *, p Ͻ 0.05 compared with control platelets; **, p Ͻ 0.05 compared with control and TRAP-stimulated platelets; both by repeated measures ANOVA.
one. In addition, there was also a trend toward synergy between 1 nM wortmannin and 10 nM S-NO-glutathione. Taken together, these results suggest that while both NO and wortmannin inhibit PI3-kinase associated with LYN and other tyrosine-phosphorylated proteins, they also act at unique sites, as well.
These functional inhibitory effects were confirmed by examining the molecular inhibitory effects of S-NO-glutathione and wortmannin on the surface expression of active GPIIb-IIIa and P-selectin, both important markers of platelet activation (25). Flow cytometric analysis shown in Fig. 9 demonstrates that both S-NO-glutathione and wortmannin significantly prevented the TRAP-induced surface expression of P-selectin and of the active conformation of GPIIb-IIIa, and that the effect of both added together was greater than either alone. DISCUSSION The major finding of this study is that NO inhibits the thrombin-induced activation of the p85/PI3-kinase pathway in platelets. Owing to the important role played by PI3-kinase in the aggregation response, this effect is likely to be one of the mechanisms underlying the antiplatelet activity of NO. While suppression of PI3-kinase activity has been shown to inhibit the production of NO in certain tissues (32), this is the first report of an effect of NO on PI3-kinase.
Although the activation of PI3-kinase in LYN signaling complexes has been demonstrated in neutrophils and other cell types, its role in platelet activation has not previously been established. In neutrophils, LYN has been thought to act as a "link" between the G protein-coupled chemoattractant receptors and the Ras/mitogen-activated protein kinase and PI3kinase pathways (9). Since the effectors and adaptors involved in thrombin-induced p85/PI3-kinase activation in the platelet are largely unknown, it is logical to propose a similar role for LYN in signal propagation from the G protein-coupled thrombin receptor. In vitro analyses have demonstrated that PI3kinase is stimulated when it associates with the SH3 domain of LYN (30) or (potentially) when its SH2 domains interact with phosphotyrosine moieties on c-CBL (31). Our data indicate that at least in some signaling complexes, the latter mechanism may be active. c-CBL was present in LYN signaling complexes, and since the quantity of the p85 subunit in LYN signaling complexes does not change with TRAP and/or NO exposure, it is highly likely that the increase in PI3-kinase activity results from direct activation of the enzyme.
One consistent and surprising finding was that TRAP induced a slight decrease in p85-immunoprecipitable (i.e. total) PI3-kinase activity despite stimulating the much smaller LYNimmunoprecipitable pool. Studies in other cell types have concluded that only a relatively small fraction of the p85/PI3kinase is activated by some agonists; thus, the activation of PI3-kinase in LYN and other positive signaling complexes may not be detectable in the total p85 immunoprecipitate. Although the mechanism by which TRAP decreases total activity is not known, it is possible that one or more downstream pathways in the platelet activation cascade may induce the serine phos-  4). B, platelet-rich plasma was incubated with inhibitors and stimulated with TRAP for 10 min as above. The TRAP incubation was carried out in the presence of monoclonal antibodies S12 and Y2/51. P-selectin expression was evaluated via flow cytometry as above. The figure shows the results from a representative experiment (n ϭ 2). phorylation of some p85 subpopulations, leading to their inactivation (4).
Protein kinase assays (Fig. 6) suggest that TRAP differentially regulates the phosphorylation of LYN and other proteins in LYN immunoprecipitates and that NO antagonizes these effects. The changes in LYN phosphorylation were not due to measurable changes in LYN protein in the cytosol because its levels remain constant, with or without S-NO-glutathione, in both the cytoplasm and cytoskeleton (data not shown). TRAP exposure led to a decrease in the in vitro phosphorylation of p53/56 LYN compared with resting platelets, while dramatically increasing the phosphorylation of an as yet unidentified 70-kDa protein. Thus, these data suggest that there must be either additional TRAP-regulatable kinases or at least one TRAP-regulatable phosphatase in LYN immunoprecipitates. These findings are consistent with the model first proposed by Sotirellis and colleagues (33) in which LYN phosphorylated at the inhibitory C-terminal tyrosine (Tyr-508) is inactive, whereas dephosphorylation at this site by an as yet unidentified phosphatase in combination with the dissociation of the C-terminal kinase CHK from the LYN signaling complex (34) leads to its activation. Further work will be required to prove that this model applies to the platelet. By whatever mechanism LYN signaling complexes are activated, the observation that NO pretreatment blocks both the positive and negative effects of TRAP on protein phosphorylation indicates that NO coordinately inhibits all of the TRAP-induced signals in the platelet that converge on LYN.
Once activated, LYN may stimulate PI3-kinase by way of the interaction between its SH3 domain and the proline-rich region of p85 or via interactions between the SH2 domains of p85 and phosphotyrosyl moieties on a third protein. Recent work has demonstrated a strong interaction between LYN and c-CBL using the yeast two-hybrid system (31) leading to a model in which LYN tyrosine phosphorylates c-CBL, which in turn activates PI3-kinase via the SH2 domains of p85. As alluded to above, this model is supported by our data. Fig. 7 clearly demonstrates that tyrosine kinase activation is necessary for the stimulation of LYN-associated PI3-kinase in platelets, whereas Fig. 4 shows that c-CBL is present in LYN signaling complexes. However, these data do not exclude the possibility that PI3-kinase is activated by way of an interaction with the p70 protein, which is markedly phosphorylated with TRAP stimulation and may itself be a LYN substrate. Shc is another candidate substrate, since it has been shown to become tyrosine-phosphorylated in LYN-PI3-kinase signaling complexes in N-formyl peptide-stimulated neutrophils (9). Whatever the precise molecular mechanism, NO's inhibition of TRAP-induced PI3-kinase may be explained by the ability of NO to maintain p53/56 LYN in a phosphorylation state similar to that observed in unstimulated platelets.
The importance of PI3-kinase in platelet function has only recently been recognized. PI3-kinase is believed to maintain the thrombin-induced activation of GPIIb-IIIa and, in turn, to participate in signaling events initiated by GPIIb-IIIa ligand binding, such as filopodial extension (1,35,36). It, thus, acts both in the "inside-out" and "outside-in" signaling pathways across this integrin receptor. The fact that the inhibitory effects of NO occur during the 1st min of TRAP stimulation indicates that NO affects the early signals of the thrombin receptor, i.e. in inside-out signaling occurring prior to completion of the GPIIb-IIIa-dependent translocation of PI3-kinase to the cytoskeleton (19).
The data demonstrating the additive inhibition of platelet aggregation, P-selectin expression, and active glycoprotein IIb-IIIa expression by NO and wortmannin are consistent with earlier reports showing the individual inhibitory effects of the two compounds (1,21). The best studied effect of nitric oxide is the stimulation of soluble guanylyl cyclase, responsible for the production of cGMP and, thus, activation of cGMP-dependent protein kinase. One effect of this serine/threonine kinase is an inhibition of the PKC/inositol phosphate pathway, as evidenced by a blunted rise in intracellular calcium and an inactivation of classical and novel protein kinase Cs (20,(37)(38)(39)(40), both of which lead to an inhibition of platelet aggregation. It is becoming increasingly apparent, however, that the PI3-kinase pathway and some PKC signaling pathways can overlap. For example, the 3-phosphorylated phosphoinositide products of PI3-kinase are able to activate a number of novel and atypical PKCs (41,42). Moreover, the phorbol ester-induced phosphorylation of pleckstrin, generally considered to be a PKC-dependent event, is wortmannin-sensitive (43). Thus, there is evidence that PI3kinase can evoke effects and responses both upstream and downstream of the PKC family taken as a whole. However, in the present study, the additivity of wortmannin and S-NOglutathione in inhibiting platelet aggregation indicates that NO and cyclic GMP-dependent protein kinase must exert their inhibitory actions at multiple sites in the platelet, not exclusively via PI3-kinase. Teleologically, such redundancy would serve to ensure that NO or NO donors effectively inhibit platelet activation regardless of the pathway of activation. The identification of other NO-modulated pathways will, therefore, be of great importance in deciphering the overall antiplatelet and antithrombotic effects of NO.