(Circulation. 2000;102:1924.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Participation of Tyrosine Phosphorylation in Cytoskeletal Reorganization, 
IIbß3 Integrin Receptor Activation, and Aspirin-Insensitive Mechanisms of Thrombin-Stimulated Human Platelets
From the Research Center and Department of Clinical Pathology (M.T.S., A.M., J.V., M.M., M.P., J.A.), University Hospital La Fe, Valencia, Spain; Division of Hematology and Medical Oncology (M.J.B., A.J.M.), Department of Medicine, VA New York Harbor Health Care System, New York, NY; and Division of Hematology and Medical Oncology (M.J.B., A.J.M.), Departments of Medicine and Pathology, Weill Medical College of Cornell University, New York, NY.
Correspondence to M. Teresa Santos, PhD, Research Center, University Hospital La Fe, Avda Campanar 21, 46009 Valencia, Spain. E-mail santos_ter{at}gva.es
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Abstract |
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Background—Fibrinogen binding to the active conformation of the
IIbß3
integrin receptor (glycoprotein IIb/IIIa) and cytoskeletal
reorganization are important events in platelet function. Tyrosine
phosphorylation of platelet proteins plays an
essential role in platelet signal transduction pathways. We studied
the participation of tyrosine kinases on these aspects of platelet
reactivity and their importance in cyclooxygenase
(COX)-1–independent mechanisms in thrombin-stimulated human
platelets.
Methods and Results—Using washed platelets from normal donors
and tyrphostin-A47 and aspirin as tyrosine kinase and COX-1
inhibitors, respectively, we found that tyrphostin-A47
downregulated (1) the thrombin-activated conformational change
of IIbß3, (2) actin polymerization and
cytoskeletal reorganization, and (3) the quantity of
tyrosine-phospho-rylated proteins associated with the
reorganized cytoskeleton. The latter are important components of
multimolecular signaling complexes. Concomitantly, platelet
aggregation and secretion were significantly reduced. Aspirin did not
affect receptor activation or tyrosine phosphorylation
but did decrease the initial (30-second) burst of actin polymerization.
Importantly, aspirin significantly amplified the inhibitory
effect of tyrphostin-A47 on all aspects of platelet reactivity that
we evaluated.
Conclusions—Tyrosine protein phosphorylation is a
regulatory control system of the inside-out mechanism of
IIbß3 activation and cytoskeletal assembly
in thrombin-stimulated human platelets. Inhibition of these aspects
of platelet function with tyrphostin-A47 is amplified when
platelets are treated with aspirin. Therefore, tyrosine
phosphorylation is a major component of early signaling
events and of COX-1–independent mechanisms of thrombin-induced
platelet reactivity. The study results may indicate a novel target
for therapeutic intervention.
Key Words: platelets • tyrosine kinases • glycoproteins • cytoskeleton • aspirin • thrombin
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The regulation of agonist-induced platelet reactivity requires coordination and control of multiple biochemical signaling pathways that culminate in platelet functional responses. These pathways include activation of GTP-binding proteins, inositol phospholipid turnover, eicosanoid metabolism, calcium movement, and protein phosphorylation processes.1 2 With regard to phosphorylation, various serine/threonine and tyrosine kinases and phosphatases are involved in biochemical events that lead to platelet activation.3 4 5
The precise role of protein tyrosine phosphorylation in signal transduction in platelets is not fully understood. However, it has functional implications. The stimulation of platelets with physiologically relevant agonists, such as collagen or thrombin, induces the phosphorylation of tyrosine residues of several platelet proteins.3 6 Conversely, the treatment of platelets with inhibitors of tyrosine kinases results in inhibition of platelet aggregation and secretion.3 4 7 8 9
Agonist-induced platelet aggregation requires fibrinogen binding to
the IIbß3 integrin
receptor and cell–cell contact to achieve ligand-mediated bridging of
IIbß3 complexes
between platelets.5 In resting platelets, the
IIbß3 receptor is
present in an inactive, nonadhesive conformation. On platelet
stimulation, a conformational change occurs, enabling binding of
adhesive proteins to the integrin. This results from an intracellular
signal transduction process known as "inside-out" receptor
signaling.5 Fibrinogen binding to the active conformation
of the receptor initiates another biochemical pathway known as
"outside-in" signaling that reinforces the platelet aggregation
process.5 The latter has been consistently
associated with tyrosine phosphorylation of
platelet proteins and is altered in Glanzman’s thrombasthenic
platelets.4 5 Less information is available concerning
the role of tyrosine kinases in the regulation of "inside-out"
mechanism or mechanisms of
IIbß3 receptor
signaling.5
Another early event after platelet activation is cytoskeletal
reorganization.10 11 12 This results in a rapid increase in
actin polymerization and the association of
IIbß3 with
actin-binding proteins (ABPs) and signaling molecules into the
platelet actin-based cytoskeleton.11 The anchorage of
IIbß3 to cytoskeletal
structures regulates adhesive properties of the receptor and stabilizes
fibrinogen binding via mechanisms that are not completely
elucidated.5 10 13 Tyrosine-phospho-rylated
proteins may play an important role in the formation of cytoskeletal
multimolecular protein complexes.14 15
Eicosanoid synthesis via cyclooxygenase-1 (COX-1) is an important pathway by which platelets respond to agonists. The inhibition of COX-1 by aspirin (ASA) is clinically beneficial for patients with vascular diseases.16 Nevertheless, platelets readily respond to stimuli by mechanisms independent of COX-1.2 17 Platelet reactivity is also modulated by cell–cell interactions that involve other blood cells, via both COX-1–dependent and –independent mechanisms.18 19 20 21 22 Mechanisms that regulate COX-1–independent pathways are of pathophysiological importance but are incompletely understood.
We evaluated the participation of tyrosine kinases in inside-out
IIbß3 receptor
activation and cytoskeletal reorganization in thrombin-stimulated
platelets. Furthermore, we determined the role of tyrosine
phosphorylation in COX-1–independent mechanisms of
platelet activation by thrombin. We used ASA for COX-1 inhibition
and tyrphostin-A47 (AG213; RG-50864) as an inhibitor of
tyrosine kinases.7 8 Tyrphostin-A47 inhibits Src, Fyn,
Yes, and Lyn tyrosine kinases and platelet aggregation and
secretion.7 8
Our data demonstrate that protein tyrosine
phosphorylation plays an important regulatory role in
the early events of stimulus–response coupling in thrombin-stimulated
platelets. This includes both inside-out mechanisms of
IIbß3 receptor
activation and cytoskeletal reorganization. Moreover, protein tyrosine
phosphorylation is a major component of ASA-insensitive
mechanisms of platelet reactivity, suggesting the possibility of a
novel pharmacological approach for the treatment of thrombotic
diatheses.
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Methods |
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Equipment and Reagents
Platelet aggregometer model 540 was obtained from Chronolog. Cell counter Microdiff 18, Epics XL flowcytometer, and Phoenix software were obtained from Coulter. The blotting system was obtained from Hoefer. Collagen was obtained from Hormon. Aprotinin was obtained from Bayer. Leupeptin, pepstatin, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Boehringer-Mannheim. Tyrphostin-A47 (AG 213, RG-50864) and tyrphostin-1 (tyrphostin-A1, AG9) were obtained from Sigma-Aldrich. Metrizamide was obtained from Nycomed. FITC-PAC-1 monoclonal antibody was obtained from Becton Dickinson. PAGE reagents and nitrocellulose membranes were obtained from Bio-Rad. [14C]5-Hydroxytryptamine (5-HT [serotonin]) creatinine sulfate, anti-mouse IgG peroxidase-linked antibody, and Hyperfilm-ECL Western blotting detection reagents were obtained from Amersham. Anti-phosphotyrosine mouse monoclonal 4G10 was obtained from Upstate Biotechnology. Protein standards were obtained from Novex. All other reagents were obtained from Sigma-Aldrich or Merck.
Blood Collection, Platelet Processing, and Labeling
Venous blood (160 mL) was obtained via free flow (after an
overnight fast) from normal volunteers who had been medication free for
15 days.18 The blood was collected into 50-mL
polypropylene tubes containing 6 mL acid-citrate-dextrose as
anticoagulant (total 46 mL)18 23 after informed consent
was obtained, as approved by the institutional review board.
Platelets were isolated, washed, and adjusted to
109 cells/mL.18 23 PRP from
acid-citrate-dextrose–anticoagulated blood was also used to prepare
14C-5-HT–labeled
platelets.21 23 For flow cytometry, platelets were
obtained before and 2 hours after the ingestion of 500 mg ASA, isolated
in a metrizamide gradient,24 and suspended in calcium-free
HEPES buffer A (5 mmol/L HEPES, 140 mmol/L NaCl, 5
mmol/L KCl, 1.2 mmol/L MgCl2, pH 7.4).
Plate Aggregation
Platelet aggregation was assessed with optical aggregometry
in 500 µL washed platelet suspension
(2x108 platelets/mL) in HEPES buffer A
containing 1 mmol/L CaCl2 and 0.38 mg/mL
fibrinogen at 37° with constant stirring (1200 rpm). Thrombin-induced
platelet aggregation was monitored for up to 5 minutes.
Tyrphostin-A47 and tyrphostin-1 (analog with similar structure but
lacking tyrosine kinase inhibitory activity) were prepared
as DMSO stock solutions (-70°C); working dilutions of
inhibitors and ASA were prepared daily in buffer.
Inhibitors were added to platelets, mixed, and
maintained at 37°C for 10 minutes without stirring before
platelet stimulation. Solvent controls (DMSO <0.1%) were run in
parallel.
5-HT Release
Imipramine (2.5 µmol/L) was added 1 minute before
stimulation to 14C-5-HT–labeled washed
platelets (2x10 platelets/mL). Aggregation was monitored for 3
minutes, and 125 µL 5x ice-cold stop solution (0.63 mol/L
formaldehyde, 0.05 mol/L EDTA) was added.25 The sample was
immediately transferred to an Eppendorf tube (0°C) and
centrifuged (13 000g, 1 minute) within 1 hour.
Supernatant [14C]5-HT was quantified through
scintillation counting.21 23
Protein Tyrosine Phosphorylation
Protein tyrosine phosphorylation was examined as
described.8 Thrombin-induced platelet aggregation was
terminated by the addition of 125 µL 5x stop solution (2% SDS,
1 mmol/L EDTA, 1 mmol/L EGTA, 0.5 mmol/L
Na3VO4, 10 mmol/L NaF,
10 mmol/L HEPES, 2 mg/mL aprotinin, 0.5 mg/mL leupeptin, 0.7 mg/mL
pepstatin, 170 mg/mL PMSF, pH 7.4) and stored at -20°C. Samples were
diluted 1:1 with 2x Laemmli’s buffer, reduced with 5%
ß-mercaptoethanol, and heated (30 minutes, 60°C). Proteins were
separated with SDS-PAGE (7.5% gels).
Separated proteins were transferred onto nitrocellulose.26 Membranes were stained with Ponceau S to determine protein transfer efficiency. Nitrocellulose was soaked (1 hour) in blocking buffer TBS (20 mmol/L Tris base, 137 mmol/L NaCl, pH 7.4) that contained 5% low-fat powdered milk and 0.05% NaN3. Membranes were washed twice (5 minutes) in TBS and incubated (2 hours, 20°C) with anti-phosphotyrosine antibody 4G10 (diluted 1:1000 in blocking buffer).8 Membranes were washed 3 times (5 minutes) with TBS and incubated with peroxidase-conjugated secondary antibody (diluted 1:3000 in blocking buffer without NaN3). After 5 washes (5 minutes) with TBS, blots were visualized with ECL chemiluminescence. Molecular weights of phosphorylated substrates were compared with known standards.
Cytoskeleton Isolation
Cytoskeleton isolation was performed essentially as
described.11 Thrombin-induced aggregation (0.5 mL;
2x108 platelets/mL) was terminated with 125
µL 5x ice-cold lysis buffer (1% Triton X-100, 5 mmol/L EGTA, 1
mg/mL leupeptin, 1 mmol/L PMSF, 1 mmol/L
Na3VO4, 50 mmol/L
Tris-HCl, pH 7.4). Detergent-insoluble fractions were pelleted
(15 600g, 4 minutes), rinsed twice with 1x lysis buffer,
resuspended in 200 µL Laemmli’s buffer, and boiled for 3 minutes.
Proteins were separated with SDS-PAGE and detected with Coomassie blue.
The presence of phosphorylated tyrosine residues in
cytoskeletal proteins was determined with
immunoblotting as described above. In these
experiments, 0.4 U/mL thrombin was used to enhance the degree of
cytoskeletal reorganization.
Flow Cytometry
Aspirin-free or ASA-treated platelets
(2x108 platelets/mL) in HEPES buffer
(10 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L KCl, 1
mmol/L MgSO4, 10 mmol/L glucose, pH 7.4)
plus 1 mmol/L CaCl2 were incubated without
stirring (10 minutes, 37°C) with tyrphostin-A47, tyrphostin-1
(negative control), or solvent. Thrombin was added, and incubation was
continued for 5 minutes without stirring. Duplicate 5-µL aliquots of
thrombin-stimulated platelets were transferred to polypropylene
tubes that contained 50 µL HEPES buffer without
calcium.27 To each sample, 4 µL FITC-PAC-1 monoclonal
antibody (25 µg/mL) was added, kept undisturbed (30 minutes, 20°C,
dark), quench-diluted with 500 µL ice-cold HEPES, and maintained at
0°C in the dark. To quantify the percentage of platelets binding
FITC-PAC-1, 5000 platelets per sample were analyzed.
Because 0.1 U thrombin/mL yielded PAC-1 binding in only 4.25% of
platelets, 1 and 0.4 U/mL concentrations were used.
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Results |
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Effects of Tyrphostin-A47 on Platelet Function and Tyrosine Phosphorylation of Proteins
Platelet aggregation and release were monitored 3 minutes after the addition of thrombin (0.1 U/mL) to ASA-free or ASA-treated platelets (same donor). Aspirin alone yielded the expected inhibition of platelet aggregation and release compared with ASA-free platelets (Table 1
).
Tyrphostin-A47 significantly inhibited platelet aggregation and
5-HT release in ASA-free platelets (Table 1), as reported
for tyrosine kinase inhibitors.3 4 8 9 When
ASA and tyrphostin-A47 were combined, a further reduction in
platelet aggregation and secretion occurred, exceeding the effect
of either ASA or tyrphostin-A47 alone (Table 1). The inactive
analog (tyrphostin-1) had no significant inhibitory effect
(Table 1). Functional effects of tyrphostin-A47 occurred
concomitant with a decrease in tyrosine-phosphorylated
substrates in thrombin-stimulated platelets (Figure 1). The effect was greater when ASA and
tyrphostin-A47 were combined (Figure 1). In contrast, the
tyrphostin-1 analog had no effect on protein tyrosine
phosphorylation of ASA-free or ASA-treated
platelets (Figure 1).
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Time course studies of platelet aggregation in response to thrombin
demonstrated a time-dependent increase in amplitude (Table 2, control). This was inhibited at all
time points with either ASA or tyrphostin-A47 alone. Interestingly,
inhibition with ASA at 30 seconds (53%) was much greater than that at
3 minutes (21%). Platelet aggregation was inhibited even more
strongly when tyrphostin-A47 was added to ASA-treated platelets,
particularly at an early time point (30 seconds, 97%) (Table 2).
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The results of kinetic studies of the effect of tyrphostin-A47 on
protein tyrosine phosphorylation in whole cell extracts
of ASA-free and ASA-treated platelets are shown in Figure 2. Tyrphostin-A47 (90 µmol/L)
markedly inhibited tyrosine phosphorylation of the
75/80-, 64-, and 50- to 60-kDa substrates and virtually blocked
phosphorylation of the 95/97-kDa proteins (Figure 2).
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Aspirin treatment alone had no effect on tyrosine
phosphorylation at any time point (Figure 2
).
Importantly, tyrphostin-A47 had a much greater inhibitory
effect on tyrosine phosphorylation when added to
ASA-treated platelets (Figure 2). Tyrphostin-A47
consistently induced phosphorylation of a
90-kDa protein. This was strongly reduced with ASA in
tyrphostin-A47–treated platelets (Figure 2).
To confirm the greater effect of tyrosine kinase inhibition in ASA-treated platelets, we also used a different tyrosine kinase inhibitor, genistein. The treatment of platelets with 100 µmol/L genistein had a greater inhibitory effect on tyrosine phosphorylation and platelet function when platelets were treated with ASA (not shown). Thus, tyrosine phosphorylation of platelet proteins is an important regulatory mechanism in COX-1–independent platelet reactivity to thrombin.
Effect of Tyrphostin-A47 on Inside-Out Activation of the
IIbß3 Integrin Receptor: Modulation
by Aspirin
The binding of FITC-PAC-1, a monoclonal antibody specific for
neoepitopes exposed on the activated form of the
IIbß3, was determined
in ASA-free and ASA-treated platelets through flow
cytometry.27 28 Tyrphostin-A47 (90 µmol/L)
significantly reduced thrombin (1 U/mL)-induced PAC-1 binding, whereas,
as reported,29 ASA alone had no effect (Figure 3). The reduction in PAC-1 binding was
significantly enhanced when ASA-treated platelets were exposed to
tyrphostin-A47. This trend was also observed with 0.4 U/mL thrombin
(Figure 3). In contrast, tyrphostin-1 did not modify PAC-1
binding.
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P<0.05).
Tyrphostin-1 (analog) did not modify PAC-1 binding in ASA-free
(mean±SEM control 80.4±2.86, tyrphostin-1 85.68±2.28) or ASA-treated
(control 72.80±6.40, tyrphostin-1 72.38±2.83; n=12) platelets.
Statistical analysis by ANOVA plus Bonferroni’s test.
Thus, tyrphostin-sensitive tyrosine kinases modulate inside-out
IIbß3 receptor
activation. Moreover, ASA-sensitive events converge with tyrosine
kinase–modulated effects to downregulate the initial integrin
activation.
Effect of Tyrphostin-A47 on Organization of the Platelet
Cytoskeleton: Role of Aspirin
We stimulated ASA-free or ASA-treated platelets with thrombin
(0.4 U/mL) in the presence or absence of tyrphostin-A47. Aspirin
treatment decreased the quantities of ABP, talin, and polymerized actin
at 30 seconds compared with control platelets (Figure 4). However, the ASA inhibition was
overcome if platelet aggregation was allowed to proceed.
Tyrphostin-A47 (90 µmol/L) significantly reduced the quantity of
polymerized actin and associated proteins in the cytoskeleton of
thrombin-stimulated platelets, particularly at early time points
(Figure 4). When platelets were simultaneously
treated with ASA and tyrphostin-A47, a greater reduction was observed
in ABP and talin in the cytoskeleton (Figure 4). Thus, tyrosine
kinase activities modulate platelet cytoskeletal reorganization,
whereas ASA downregulates the initial burst of actin polymerization and
augments the inhibitory effects of tyrphostin-A47.
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Effect of Aspirin and Tyrphostin-A47 on Tyrosine
Phosphorylated Proteins on the Cytoskeleton
Shortly after thrombin stimulation,
phosphorylation of 130-, 75/80-, 64-, and 50/60-kDa
proteins was detected in the cytoskeleton. The 95/97-kDa substrates
appeared later (3 minutes) (Figure 5).
The pattern of tyrosine-phosphorylated substrates in
the cytoskeleton was similar, although not identical, to that observed
in whole cell extracts (Figure 2 versus Figure 5).
Tyrosine-phosphorylated proteins in the cytoskeleton
were minimally detectable in unstimulated platelets (not
shown).
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Aspirin treatment of platelets significantly reduced the quantity
of tyrosine-phosphorylated proteins in the cytoskeleton
at 30 seconds (Figure 5). This was in sharp contrast with the
lack of effect of ASA on tyrosine phosphorylation in
whole cell extracts (Figure 2). No differences were observed
between control and ASA-treated platelets at later time points
(Figure 5).
Tyrphostin-treatment (90 µmol/L) of ASA-free platelets
markedly decreased tyrosine-phosphorylated proteins in
the cytoskeleton at 30 seconds and 3 minutes. However, at 5 minutes,
the differences observed between tyrphostin-treated and control
platelets were minimal (Figure 5). Importantly,
tyrphostin-A47 virtually abolished the presence of
tyrosine-phosphorylated proteins in the cytoskeleton at
all time points in ASA-treated platelets (Figure 5).
Because tyrosine-phosphorylated motifs are important for the assembly of multimolecular signaling complexes in the cytoskeleton,14 15 this suggests that ASA plus tyrphostin-A47 is a potent inhibitor of this process.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Fibrinogen binding to the activated
IIbß3 integrin
receptor and cytoskeletal reorganization are important events in
platelet function. The reorganized cytoskeleton serves as a
scaffold for association of cytoskeletal proteins, integrins, and
signaling molecules.14 The resulting multimolecular
complexes participate in signal transduction and platelet
functional responses.3 10 Tyrosine
phosphorylation of platelet proteins is an
essential component of platelet signal transduction
pathways.4 5 We studied the participation of tyrosine
kinases on these aspects of platelet reactivity and investigated
their importance in COX-1–independent mechanisms in
thrombin-stimulated human platelets. COX-1–independent mechanisms
are of importance because clinical trials indicate that only 25% to
30% of patients with vascular disease benefit from ASA
therapy.16 We hypothesize that the lack of benefit in the
remaining 70% of patients is due, at least in part, to platelet
activation by mechanisms independent of COX-1. This also includes
stimulatory effects elicited by cell–cell interactions with other
blood cells as we and others have demonstrated.18 19 20 21 22
Our results extend previous data on the inhibition of platelet
aggregation and secretion with tyrosine kinase
inhibitors.3 4 8 9 Importantly, this
inhibitory effect is significantly increased in ASA-treated
platelets (Tables 1 and 2). Thus, tyrosine
phosphorylation constitutes a major COX-1–independent
mechanism whereby platelets respond to thrombin. The effects of
tyrphostin-A47 are due to tyrosine kinase inhibition specifically,
because tyrphostin-1 (negative control) is inactive (Table 1,
Figure 1). Moreover, the amplified inhibitory effect
with ASA was also observed with genistein (another kinase
inhibitor).
Regarding the biochemical mechanisms that underlie these
functional effects, our results show that tyrosine kinases modulate
(directly and/or indirectly) inside-out signaling of
IIbß3 in
thrombin-stimulated platelets. This follows from our observation
that tyrphostin-A47 significantly reduces thrombin-induced PAC-1
binding (Figure 3). This is in agreement with data with
genistein-treated permeabilized
platelets,28 platelets stimulated with the
thrombin receptor agonist peptide SFLLRN,29
piceatannol-treated ADP-stimulated platelets,30 and
platelets treated with herbimycin A.9 The specific
kinases involved have not been identified. Law et al30
reported the participation of Syk tyrosine kinase, because receptor
activation is inhibited (32%) in Syk-null murine platelets.
However, this partial inhibition suggests that tyrosine kinases other
than Syk are also involved in this step of receptor activation.
Although ASA alone did not modify receptor activation, as
reported,29 the inhibition of tyrosine kinase in
ASA-treated platelets resulted in further reduction in receptor
activation as shown by reduced PAC-1 binding (Figure 3). This
demonstrates ASA amplification of tyrosine kinase inhibition of this
important aspect of platelet reactivity and suggests that
IIbß3 downregulation
is at least 1 mechanism whereby tyrosine kinases regulate
COX-1–dependent and –independent mechanisms of platelet
reactivity.
Our results show that tyrosine kinase inhibition with tyrphostin-A47
reduced actin polymerization and decreased the quantity of
actin-associated proteins in cytoskeletons of thrombin-stimulated
platelets, particularly at early time points (Figure 4).
This demonstrates that tyrosine kinases regulate cytoskeletal
reorganization. Unexpectedly, we consistently observed an
ASA-sensitive, but reversible, decrease in cytoskeletal reorganization
shortly after thrombin addition (30 seconds) (Figure 4). Thus,
an ASA-sensitive mechanism regulates the early burst of actin
polymerization. When ASA- and tyrphostin-sensitive mechanisms were
inhibited simultaneously, greater inhibition of
cytoskeletal reorganization resulted. This represents a novel,
COX-1–independent mechanism of tyrosine kinase regulation of
platelet function.
In our experiments, tyrphostin-A47 effects on
IIbß3 activation and
cytoskeletal reorganization paralleled a reduction in
tyrosine-phosphorylated proteins in the actin-based
cytoskeleton (Figure 5). Interestingly, ASA also reduced
tyrosine phosphorylation of proteins in the
cytoskeleton shortly after thrombin addition (30 seconds) (Figure 5). This coincides temporally with decreased actin
polymerization (Figure 4) and the greatest degree of inhibition
of platelet aggregation (Table 2). It is unrelated to an
effect of ASA on tyrosine kinase activity, because ASA had no effect on
tyrosine-phosphorylated proteins in whole cell extracts
(Figure 2). Importantly, tyrphostin-A47 induced a dramatic
reduction of tyrosine-phosphorylated proteins in the
cytoskeleton of ASA-treated platelets (Figure 5). This is of
interest if we consider that protein phosphotyrosine domains and SH2
and SH3 homology regions are essential components of protein–protein
assembly for multimolecular complex formation in the
cytoskeleton.14 These protein complexes participate in
focal adhesion, signal transduction, and platelet functional
responses.3 10 Therefore, the inhibition demonstrated in
our study of tyrosine-phosphorylated proteins in the
cytoskeleton might represent a reduction in signaling complex
formation in the cytoskeleton of thrombin-stimulated human
platelets. This would contribute to the significant inhibition of
platelet aggregation (Tables 1 and 2),
IIbß3 receptor
activation (Figure 3), and cytoskeletal reorganization (Figure 4) that we observed.
In conclusion, our results support the concept that tyrosine
kinases participate in inside-out mechanisms of
IIbß3 receptor
activation and extend it to the amplifying effect of ASA on
downregulation that results from tyrosine kinase inhibition. In
addition, we present novel evidence of regulation by tyrosine
kinases of both cytoskeletal organization of structural proteins and
the accumulation of tyrosine-phosphorylated proteins in
actin-rich cytoskeleton of thrombin-stimulated platelets. These are
required for multimolecular signaling complex formation in the
cytoskeleton. Furthermore, our ASA experiments demonstrate that
tyrosine phosphorylation is a major component of
COX-1–independent mechanisms of platelet activation with thrombin.
This may constitute a novel target for future antithrombotic drug
development.
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Acknowledgments |
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This work was supported in part by grants 95/0639 and 98/0906 from the Fondo de Investigaciones Sanitarias (FIS) (Drs Santos, Vallés, and Aznar); a Merit Review grant from the Department of Veterans Affairs (Dr Marcus); and NIH grants HL-47073, HL-46403, and HL-07423 (Drs Marcus and Broekman). The authors gratefully acknowledge the technical assistance of M. Carmen Insa, Amparo Garrido, and Pilar Ferriz.
Received February 25, 2000; revision received May 23, 2000; accepted May 31, 2000.
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