From the Whitaker Cardiovascular Institute and Evans
Department of Medicine, Boston University School of Medicine,
Boston, Massachusetts 02118, the ** Department of Pediatrics,
University of Massachusetts Medical School,
Worcester, Massachusetts 01605, and ¶ Unit for Metabolic
Diseases, HS Raffaele, via Olgettina 60, Milano, Italy
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 donor
S-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
Exposure of platelets to thrombin or the thrombin receptor-activating
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- Chemicals and Solutions--
Wortmannin, sodium nitrite, bovine
serum albumin, hydrochloric acid, Sepharose 2B, HEPES, Triton X-100,
EDTA, sodium pyrophosphate, NaF, aprotinin, phenylmethylsulfonyl
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.
[ Antibodies--
Polyclonal antibodies against LYN and p85 and
monoclonal antiphosphotyrosine antibodies were purchased from Upstate
Biotechnology (Lake Placid, NY). Monoclonal anti-LYN antibody was
obtained from Transduction Laboratories (Milwuakee, WI). Fluorescein
isothiocyanate (FITC)-conjugated monoclonal antibody PAC1 was purchased
from Becton Dickinson Immunocytometry Systems (San Jose, CA).
Phycoerythrin-conjugated monoclonal antibody P2 was purchased from
Immunotech (Westbrook, ME). Biotinylated monoclonal antibody S12 was
obtained from Centocor (Malvern, PA). FITC-conjugated monoclonal
antibody Y2/51 was purchased from Dako Corp. (Carpinteria, CA).
Monoclonal antibody raised against GLUT4 (clone IF8) was a gift from
Dr. Paul Pilch of Boston University.
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 × 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 × 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 2× 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 × 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 µl 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 µl of 1× 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 µl of 25 mM HEPES, 100 mM NaCl, 1 mM sodium orthovanadate, 3 mM MnCl2, 10 mM MgCl2,
0.5 µM ATP, and 20 µCi of [
For LYN kinase assays using histone H1 as a substrate in LYN
immunoprecipitates, 30 µl of the foregoing kinase reaction mixture containing 50 µg/µl 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 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 µl of 10 mM Tris, pH 7.5, and 1 mM EGTA. This PI mix was added to each tube together with
50 µl of the third washing buffer described above and 10 µl of 100 mM MgCl2. The reaction was then initiated with
the addition of 5 µl of a solution composed of 20 mM
MgCl2, 0.88 mM ATP, and 2 µCi of
[ Platelet Aggregation Assay--
Platelet aggregation was
monitored by standard nephelometric techniques (24) using a BioData
aggregometer (BioData, Inc., Hatsboro, PA).
Flow Cytometry--
Two-color flow cytometric analysis was used
to determine the activation of platelet surface glycoprotein IIb-IIIa
(the fibrinogen receptor integrin IIb
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 fibrinogen-binding 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 ± 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 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 µM) 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 + 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 (27-29). 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 + 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 + 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-NO-glutathione, 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-NO-glutathione (TRAP + S-NO) for 10 min and then stimulated with TRAP. Densitometry showed that the amount
of PI3-kinase 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) indicated
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 signaling 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 LYN-associated PI3-kinase
activity is that LYN becomes activated by upstream signaling elements
and then itself activates PI3-kinase. 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 + 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 + S-NO). Interestingly, p53/p56 LYN was less
phosphorylated in TRAP-stimulated than in control platelets, whereas
S-NO-glutathione 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-NO-glutathione
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 µM) 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. Genistein 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 <100 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 µM; S-NO-glutathione, 0.1 nM to 10 µM). Fig.
8 shows that wortmannin inhibited
TRAP-induced aggregation by over 80% at 100 nM and that its IC50 was 10 nM. For S-NO-glutathione, there
was nearly complete inhibition of aggregation by concentrations as low
as 1 µM, and its IC50 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-glutathione. 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.
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 PI3-kinase 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
PI3-kinase 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 LYN-immunoprecipitable pool. Studies in other cell types have concluded that only a relatively small fraction of the p85/PI3-kinase 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 phosphorylation 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, 37-40),
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 PI3-kinase 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-NO-glutathione 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
, 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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]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.
-32P]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.
volume of 3× loading buffer,
and the phosphorylated proteins were resolved by SDS-PAGE and
visualized by autoradiography.
-32P]ATP. Incubations were carried out for 10 min and
stopped with the addition 20 µl of 8 M HCl and 160 µl
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.
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 µM S-NO-glutathione for 10 min or both S-NO-glutathione
and wortmannin. Subsequently, 15 µl 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 µg/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.
0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 1.
PI3-kinase activity in phosphotyrosine, LYN,
and p85 immunoprecipitates. A, gel-filtered platelets
(500 µl) were stimulated with 10 µM TRAP for 1 min
after a 10-min preincubation with 10 µM 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 (±S.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 (±S.E.). *,
p < 0.05 compared with control platelets; **,
p < 0.05 compared with TRAP-stimulated platelets; each
by repeated measures ANOVA.
View larger version (27K):
[in a new window]
Fig. 2.
Physical association between LYN and
PI3-kinase. Immunoprecipitations were carried out with anti-LYN
monoclonal antibodies using Triton-soluble lysates from resting
platelets (CON) or platelets stimulated with 10 µM TRAP for 1 min after being incubated for 10 min in the
absence (TRAP) or presence of 10 µM
S-NO-glutathione (TRAP + S-NO). After the
immunoprecipitation, proteins were separated by SDS-PAGE and
transferred to nitrocellulose, and Western blotting was carried out
with polyclonal anti-p85. Shown are blots on duplicate lanes under each
of the three conditions.
View larger version (44K):
[in a new window]
Fig. 3.
Size of p85-associated LYN pool and
LYN-associated p85 pool. Gel-filtered platelets were preincubated
with or without 10 µM TRAP for 1 min and with or without
S-NO-glutathione (S-NO) for 10 min and solubilized as in
Fig. 2. Total protein samples were taken before and after
immunoprecipitation (IP) with anti-LYN (A) and
anti-p85 antibodies (B), and the proteins were separated by
SDS-PAGE. Western blots were carried out with anti-p85 (top
row in each panel) and anti-LYN (bottom row)
antibodies. The blots shown are representative from three
experiments.
View larger version (22K):
[in a new window]
Fig. 4.
c-CBL in LYN/PI3-kinase signaling
complexes. Platelets were incubated with vehicle ( ) or TRAP (+).
Duplicate Triton-soluble lysates (1 ml each) were then
immunoprecipitated with monoclonal anti-LYN, and the proteins were
separated by SDS-PAGE as in Fig. 2. Three hundred µl (1st
lane in each pair) or 900 µl (2nd lane in each pair)
of lysate was used for the immunoprecipitation (IP) with 2 µg of antibody. Aliquots of the parent extract equivalent to 15 µl
(1st lane in each pair) or 50 µl (2nd lane in
each pair) of the total were also subjected to SDS-PAGE. Western
blotting was then carried with polyclonal anti-CBL antibody.
View larger version (29K):
[in a new window]
Fig. 5.
Effect of 8-Br-cGMP on TRAP-stimulated
PI3-kinase. 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 (±S.E.). *,
p < 0.05 compared with control platelets; **,
p < 0.05 compared with TRAP-stimulated platelets; each
by repeated measures ANOVA.
View larger version (51K):
[in a new window]
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 µM TRAP for 1 min (TRAP), or platelets
preincubated with 10 µM S-NO-glutathione for 10 min prior
to TRAP stimulation (TRAP + 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 = 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 = 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.
View larger version (33K):
[in a new window]
Fig. 7.
Effect of genistein and PP2 on the
stimulation of TRAP of the LYN-associated PI3-kinase.
A, gel-filtered platelets were preincubated with buffer
(CON) or 5 µM genistein (GEN) for
30 min; some samples were then stimulated with 10 µM TRAP
for 1 min (TRAP and TRAP + GEN, respectively).
B, similar experiments were carried out using the SRC family
kinase inhibitor PP2 (5 µM). 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
(±S.E.). *, p < 0.05 compared with control platelets;
**, p < 0.05 compared with control and TRAP-stimulated
platelets; both by repeated measures ANOVA.
View larger version (22K):
[in a new window]
Fig. 8.
Combined platelet inhibitory effect of
wortmannin and S-NO-glutathione. A, gel-filtered
platelets were preincubated with the indicated concentrations of
wortmannin for 30 min or with the indicated concentrations of
S-NO-glutathione for 10 min prior to being stimulated with TRAP for 2 min, during which time aggregation was monitored. Control aggregations
were determined in the absence of inhibitors within each run, and the
results were normalized to that value. In absolute terms, the control
aggregations ranged in extent from 50 to 80%. The experiment was
repeated 3-4 times for the highest two concentrations of inhibitors
and 11-15 times for other concentrations. B, in some
experiments the indicated combinations of inhibitors were added.
Results were analyzed by repeated measures ANOVA (n = 4 for each of the 3 combinations); *, p < 0.05 relative
to wortmannin (WM); **, p < 0.05 relative
to both wortmannin and S-NO-glutathione.
View larger version (29K):
[in a new window]
Fig. 9.
Combined effect of wortmannin and
S-NO-glutathione in inhibiting the platelet surface expression of
GPIIb/IIa and P-selectin. A, platelet-rich plasma was
preincubated with 100 nM wortmannin (WM) for 30 min, 10 µM S-NO-glutathione (S-NO) for 10 min,
or both (WM + NO), and then with PAC1 antibody, P2 antibody,
and TRAP for 10 min. The platelets were fixed in 4% formaldehyde and
analyzed via flow cytometry for GPIIb-IIIa activation as described
under "Experimental Procedures." The figure shows the results from
a representative experiment (n = 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 = 2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank C. R. Kahn, Joslin Diabetes Center, Boston, for help and discussion on preliminary experiments. We also thank Stephanie Tribuna and Michael Hollywood for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL48976, HL53919, HL55993, and HL48743 and by a Merit Review Award from the Dept. of Veterans Affairs.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.
§ Both authors contributed equally to this work.
Supported by grants from H. S. Raffaele, Milano, Italy
and Ministero della Sanitá, Rome, Italy.
To whom correspondence should be addressed: Boston University
School of Medicine, Whitaker Cardiovascular Institute, 700 Albany St.,
Boston, MA 02118. Tel.: 617-638-4890; Fax: 617-638-4066; E-mail:
jloscalz{at}bu.edu.
2 G. M. Bokoch, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NO, nitric oxide; PI3-kinase, phosphoinositide 3-kinase; S-NO-glutathione, S-nitrosoglutathione; TRAP, thrombin receptor-activating peptide; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PKC, protein kinase C; ANOVA, analysis of variance; PI, phosphatidylinositol; GP, glycoprotein; SH, SRC homology.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|