© 2000 American Heart Association, Inc.
Thrombosis |
Inhibition of Platelet Integrin 
IIbß3 by Peptides That Interfere With Protein Kinases and the ß3 Tail
From the Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht and Institute for Biomembranes, Utrecht University, Utrecht, the Netherlands. Dr Hers is now at the Department of Biochemistry, School of Medical Sciences, Bristol, UK.
Correspondence to Prof Dr J.W.N. Akkerman, Department of Haematology, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, Netherlands. E-mail j.w.n.akkerman{at}laboratory.azu.nl
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Abstract |
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Abstract—
-Thrombin
stimulation of human platelets initiates inside-out signaling to
integrin IIbß3 (glycoprotein
IIb/IIIa), resulting in the exposure of ligand binding sites. In the
present study, the regulation of IIbß3
via protein kinases was investigated in platelets
permeabilized with streptolysin O by introducing
peptides that interfere with these enzymes and with possible regulatory
domains in the cytosolic tail of the ß3 subunit. Compared
with intact platelets, the permeabilized
platelets preserved >80% of the aggregation, secretion, and
IIbß3 ligand binding capacity. The peptide
YIYGSFK, a substrate for Src kinases, inhibited -thrombin–induced
ligand binding to IIbß3, but a reversed
peptide with Y
F substitutions (KFSGFIF) had no effect. Ligand
binding to IIbß3 was also inhibited by the
peptide RKRCLRRL, which binds irreversibly to the catalytic domain of
protein kinase C. Peptides corresponding to parts of the protein C
inhibitor and ß2-glycoprotein I
were used as negative controls and failed to interfere with ligand
binding. Possible target domains for protein kinases are present in
the cytoplasmic tail of the ß3 subunit. The LLITIHDR
peptide, matching the membrane-proximal domain of ß3
(residues 717 to 724), had no effect, but NNPLYKEA (residues 743 to
750), EATSTFTN (residues 749 to 756), and TNITYRGT (residues 755 to
762), which mimicked overlapping domains of the carboxy-terminal part
of ß3, reduced -thrombin–induced ligand binding by
60±4%, 97±1%, and 97±2% (n=3) at 500 µmol/L peptide,
respectively. These observations indicate that Src kinases and protein
kinase C take part in inside-out signaling to integrin
IIbß3 and identify target domains in
ß3 that contribute to the regulation of this
integrin.
Key Words: integrin IIbß3 • glycoprotein IIb/IIIa • protein kinase C • protein tyrosine kinase • platelets
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Integrins are heterodimeric cell surface receptors consisting of noncovalently associated
and ß
subunits.1 Each subunit contains a large extracellular
domain, a transmembrane domain, and a relatively short cytoplasmic tail
(typically <70 residues for each subunit). Integrins function in a
variety of biological processes, such as the differentiation, growth,
and migration of cells, and in inflammation and wound healing. Many
integrins are subject to intracellular modulation of their activation
state for ligands, a process known as inside-out
signaling.2 3 4 An increase in binding affinity and
subsequent ligand binding generates signals into the cell, a process
known as outside-in signaling.5 6 7 8
The regulation of platelet integrin
IIbß3 is a good
illustration of the significance of the rapid affinity regulation of
integrins. Unstimulated platelets express
IIbß3 in a
conformation inaccessible to ligands, thereby preventing
platelet-platelet interaction. On activation, inside-out
signaling converts the integrin to a functional receptor for
fibrinogen, fibronectin, von Willebrand factor, and
vitronectin.9 Fibrinogen binding to
IIbß3 couples the
platelets together, forming an aggregate, which is a vital step in
the cessation of bleeding. Patients with Glanzmann’s
thrombasthenia, a severe bleeding disorder, have platelets that
either lack IIbß3
(type I) or contain a defective
IIbß3 that is unable
to expose ligand binding sites (type III).10 The
Ser752Pro mutation in the ß3 subunit of type
III patients is of particular interest because it results in the loss
of ligand binding.11
Platelet agonists trigger inside-out signaling to
IIbß3 via protein
tyrosine kinases (PTKs) as herbimycin A, tyrphostin A47, and
geldanamycin inhibit ligand binding.12 The tyrosine
phosphorylation of proteins with molecular masses of
54, 60, 64, 75, and 130 kDa during binding site exposure suggests that
they may contribute to the affinity regulation of
IIbß3.13 14 15
Only a few agonists signal to IIbß3
via PTK and protein kinase C (PKC, a Ser/Thr kinase), as illustrated by
the inhibition by bisindolylmaleimide I and II and calphostin C and
also by the fact that phorbol 12-myrisate 13-acetate (an
activator of the PKC family) and
12-deoxyphorbol-13-phenylacetate-20-acetate (an activator
of the PKCß subtype) trigger the sustained exposure of ligand binding
sites. Activated PKC contributes to PTK activation and
preserves the high-affinity state of the integrin, probably via
stoichiometric phosphorylation of the
ß3 subunit.16 Although kinase
inhibitors have provided a first insight into the signaling
pathways that control platelet
IIbß3, their broad
specificity and the concern about their effects on other steps in
platelet activation are obvious limitations. In the present
study, we have applied specific peptides designed to interfere with the
activity of these kinases and the accessibility of their possible
target domains in
IIbß3. These peptides
were (1) YIYGSFK, a substrate peptide for the Src-family; (2) RKRCLRRL,
a peptide that binds irreversibly to the catalytic domain of PKC; and
(3) 4 peptides mimicking domains in the cytoplasmic tail of the
ß3 subunit containing potential
phosphorylation sites for PTKs and PKCs.
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Methods |
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Materials
Streptolysin O (SLO) was obtained from Murex diagnostics. GDPßS, GTP
S, D-myo-inositol
1,4,5-tris-phosphate (IP3), bisindolylmaleimide I
(GF 109203X), and ADP were purchased from Roche Molecular Biochemicals.
-Thrombin, imipramine, and peroxidase-labeled protein A were
obtained from Sigma Chemical Co, and Sepharose 2B and gelatin Sepharose
4B were from Pharmacia. Sodium iodide (specific activity 629
GBq/mg) and [14C]serotonin
(specific activity 1.8 to 2.2 GBq/mmol) were purchased from Amersham
International. [32P]Orthophosphate with a
specific activity of 314 TBq/mmol was from New England Nuclear.
D-Phenylalanyl-L-prolyl-L-arginine
chloromethylketone (PPACK) was from Calbiochem. The
anti-phosphotyrosine antibodies PY-20 and 4G10 were from Upstate
Biotechnology Inc. Sulforhodamine B was from Molecular Probes Inc.
Fibrinogen (grade L) was from Chromogenix, and the blocking
anti–integrin ß1 subunit antibody AIIB2 was a
kind gift from Dr C Damsky, University of California, San
Francisco.17 The fibrinogen-derived peptide GRGDS was
kindly provided by Dr Bekkers, Department of Enzymology and Protein
Engineering, Utrecht University, Utrecht, the
Netherlands. The polyclonal anti-Syk antibody SC-573 was from Santa
Cruz Biotechnology Inc, and the swine anti-rabbit horseradish
peroxidase–labeled antibody was from DAKO. Other chemicals were of
analytical grade.
Peptides
Peptides were synthesized by a stepwise solid-phase peptide
synthesis method and purified by C18 reverse-phase
high-performance liquid chromatography (HPLC,
Biosynthesis). The purity of the peptides was >99%. The molecular
weights of the peptides were verified by matrix-assisted laser
desorption mass spectrometry by the manufacturer. The synthesized
peptides were an Src substrate peptide (Src peptide, YIYGSFK, 7 amino
acids [AAs], molecular weight [MW] 877), the reversed Src peptide
with YF substitutions (Src-R peptide, KFSGFIF, 7 AAs, MW 845), the
inhibitory peptide for PKC (PKC peptide, RKRCLRRL, 8 AAs,
MW 1100), and 4 peptides corresponding to different partially
overlapping domains of the ß3 cytoplasmic tail
(shown in Figure 1
). These 4 peptides
were as follows: ß3-1 (corresponding to
residues 717 to 724, 8 AAs, MW 980), ß3-2
(residues 743 to 750, 8 AAs, MW 918), ß3-3
(residues 749 to 756, 8 AAs, MW 870), and ß3-4
(residues 755 to 762, 8 AAs, MW 925). In addition, 2 modifications of
the ß3-3 peptide were synthesized with an SA
(EATATFTN) and an SP replacement (EATPTFTN). For uptake studies, the
ß3-3 and ß3-4 peptides
were labeled with FITC by dissolving 1 mg peptide in 300 µL of 0.25
mol/L sodium carbonate buffer (pH 9.0) and incubated with 1.3
mmol/L FITC for 16 hours at 4°C. The FITC-labeled peptide was
purified on a C8 reverse-phase HPLC column. The molecular mass of the
purified peptide was verified by mass spectrometry. To assess the
aspecific effects after the introduction of peptides, platelets
were treated with protein C inhibitor (PCI)-1 peptide
(HRHHPREHKERVEDLH, 16 AAs, MW 2112), PCI-2 peptide (HRHHPREHKEEVEDLH,
16 AAs, MW 2085), and
ß2-glycoprotein I (ß2-gpI)
peptide (FKEHSSLAFWK, 11 AAs, MW 1402), which were >96% pure and
kindly provided by Drs M.G.L.M. Elisen and D.A. Horbach, Department of
Hematology, University Medical Center Utrecht,
Utrecht, the Netherlands. The PCI peptides correspond to a
domain in the heparin binding site of PCI and contain individual
substitutions of a basic residue in the consensus binding domain that
blocks heparin binding. The ß2-gpI peptide corresponds to a sequence
in the fifth domain of ß2-gpI.
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Platelet Isolation
Freshly drawn venous blood from healthy volunteers, who claimed
not have taken any medication in the previous 14 days, was
anticoagulated with 0.1 vol of 13 mmol/L trisodium citrate.
Platelet-rich plasma was obtained by centrifugation
(200g for 15 minutes at 22°C), and platelets were
isolated by gel filtration on a Sepharose 2B column equilibrated in
Ca2+-free Tyrode’s solution (137 mmol/L
NaCl, 2.68 mmol/L KCl, 0.42 mmol/L
NaH2PO4, 1.7 mmol/L
MgCl2, and 11.9 mmol/L
NaHCO3, pH 7.2) containing 0.1% glucose and
0.2% BSA. The platelet count was adjusted to
2x1011 platelets per liter.
Preparation of 125I-Labeled Fibronectin and
125I-Labeled Fibrinogen
Fibronectin was isolated from fresh frozen plasma by affinity
chromatography on gelatin–Sepharose 4B as described
elsewhere.18 Electrophoresis on a 5%
polyacrylamide gel, as described by Laemmli19
after reduction with ß-mercaptoethanol, showed >99% homogeneity.
The fibronectin preparation contained <10 ng von Willebrand
factor per milligram of fibronectin, as determined with a von
Willebrand ELISA. Fibrinogen was made fibrin and fibronectin
free by passage through a gelatin–Sepharose 4B column. The fibrinogen
and fibronectin were radiolabeled with sodium iodide by a modified
Iodo-Gen method, as described in detail for fibrinogen.20
Isolation and Permeabilization With SLO
A 15 U/mL solution of SLO was freshly prepared in
Ca2+-free Tyrode’s solution (137 mmol/L
NaCl, 2.68 mmol/L KCl, 0.42 mmol/L
NaH2PO4, 1.7 mmol/L
MgCl2, and 11.9 mmol/L
NaHCO3, pH 7.2) containing 0.1% glucose and
0.2% BSA. Gel-filtered platelets were
permeabilized with 0.15 U/mL SLO for 3 minutes at
22°C before addition of the agonist, unless indicated otherwise. In
each experiment, permeabilization was verified by a parallel incubation
with the membrane-impermeant mediator IP3
(15 µmol/L). Suspensions that showed an
IP3-induced ligand binding of 
22 500
fibronectin molecules per platelet were considered permeable and
used for further analysis. IP3-induced
phosphorylation of pleckstrin, a substrate for
PKC, was >20% of the phosphorylation seen in the
presence of 0.2 U/mL -thrombin, as assessed by the program
ImageQuant (Molecular Dynamics). Tyrosine
phosphorylation initiated by IP3
was in the same range as that induced by the weak agonist ADP.
Experiments in which IP3 induced lower responses
were discarded. Permeabilization was confirmed by
fluorescence-activated cell sorter (FACS)
analysis in the FL-2 channel on a FACScan (Becton-Dickinson) in
the presence of sulforhodamine B. The permeabilization procedure
induced 16±4% (n=4) release of maximal secretable
[14C]serotonin (5 U/mL
-thrombin, 20 minutes at 22°C) in the course of the experiment.
[14C]Serotonin secretion was
determined as described.21
Binding of 125I-Labeled Fibronectin and
125I-Labeled Fibrinogen
Gel-filtered platelets were permeabilized in
the presence of different concentrations of the Src, PKC, and
ß3 peptides at 22°C.
125I-labeled ligand (1 µmol/L) was added 2
minutes after the addition of SLO, which was followed by the addition
of -thrombin (0.2 U/mL) or IP3 (15
µmol/L, all final concentrations) 3 minutes later. Samples (200 µL,
in triplicate) were drawn and layered on top of 100 µL of 20%
(wt/vol) sucrose in Ca2+-free Tyrode’s solution
in microsedimentation tubes (Sarstedt) and centrifuged
(12 000g for 4 minutes at 22°C) in a Beckman Microfuge E
15 minutes after stimulation. The pellet was separated from the
supernatant and counted for radioactivity in a gamma counter. The
number of molecules bound per platelet was calculated from the
radioactivity in the pellet fraction and compared with the total
activity in the pellet plus supernatant. The data were corrected
for nonspecific binding, defined as the binding of
125I-labeled ligand to unstimulated intact
platelets.18 22
Measurement of Pleckstrin Phosphorylation
The activity of PKC was deduced from the
phosphorylation of the 47-kDa protein pleckstrin, a
major substrate for this enzyme. Platelets were labeled with 3.7
MBq carrier-free [32P]Pi per milliliter of
platelet-rich plasma for 1 hour at 37°C. Platelet suspensions
were acidified to pH 6.5, centrifuged, and resuspended in
HEPES-Tyrode (145 mmol/L NaCl, 5 mmol/L KCl, 0.5 mmol/L
Na2HPO4, 1 mmol/L
MgSO4, and 10 mmol/L HEPES, pH 7.2)
containing 0.1% glucose. Labeled platelets
(2x1011 platelets per liter) were
permeabilized in the presence of different
concentrations of the Src, PKC, and ß3 peptides
at 22°C and incubated with -thrombin (0.2 U/mL) or
IP3 (15 µmol/L) for 2 minutes. Samples
were collected and transferred into 3x concentrated Laemmli sample
buffer19 and boiled for 5 minutes before SDS-PAGE (11%).
Gels were stained with Coomassie brilliant blue, and radioactive bands
were visualized by autoradiography and exposure to a
phosphoimager screen. For determination of the radioactivity of
pleckstrin, the density of the 47-kDa band was determined by the
program OptiQuant (Packard Instruments).
Measurement of Protein Tyrosine Phosphorylation
Gel-filtered platelets (2x1011
platelets per liter) were permeabilized in the
presence of different concentrations of the Src, PKC, and
ß3 peptides at 22°C. Subsequently,
platelets were incubated with -thrombin (0.2 U/mL) or
IP3 (15 µmol/L) for 2 minutes. Samples
were withdrawn, and 3x concentrated Laemmli sample buffer was added.
The samples were heated at 95°C for 5 minutes, and proteins were
separated on SDS/7.5% PAGE and electrophoretically transferred (1 hour
at 100 V) to nitrocellulose membrane in 25 mmol/L Tris/192
mmol/L glycine (pH 8.3) and 20% methanol (vol/vol) by using a
minitransblot system (Bio-Rad). The blots were blocked with
Tris-buffered saline (TBS) containing 4% BSA for 1 hour at room
temperature and subsequently incubated (16 hours at 4°C) with the
anti-phosphotyrosine antibody 4G10. Subsequently, the blots were
incubated with peroxidase-labeled protein A (2 µg/mL for 2 hours at
4°C), treated with Renaissance chemiluminescence Western blot
reagent, and exposed to Kodak X-Omat Blue
autoradiography film (Eastman Kodak Co). For the
reprobing with an anti-Syk antibody, the blots were stripped in TBS
containing 0.1% Tween 20, 2% SDS, and 2% ß-mercaptoethanol. After
extensive washing, the blots were blocked with 10% BSA in TBS-Tween
and incubated for 16 hours with a polyclonal antibody against Syk.
Subsequently, the blots were incubated with swine anti-rabbit
horseradish peroxidase–labeled antibody, and the densities of the
spots were quantified with OptiQuant (Packard Instruments).
Statistics
Data are expressed as mean±SD, with n indicating the number of
observations, and were analyzed by Student t test
for paired observations. Differences were considered significant
at P<0.05.
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Results |
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Integrin
IIbß3 Is Preserved in Human
Platelets Permeabilized by SLOPlatelets were permeabilized by treatment with SLO. The optimal SLO concentration was determined by incubation in the presence of the fluorescent indicator sulforhodamine B and by monitoring the uptake of the dye by flow cytometry. Uptake was half maximal at
0.13 U/mL and maximal at 0.20 U/mL SLO (Figure 2A). The cell-impermeable
activator IP3, which releases
Ca2+ from intracellular stores, and GTPS,
which activates trimeric G proteins, induced ligand binding of
22 500±4100 and 17 230±3280 fibronectin molecules per platelet,
respectively, at 0.15 U/mL SLO, illustrating that inside-out signaling
mechanisms to IIbß3
were at least partly preserved. Above this SLO concentration, the
functional properties of
IIbß3 were lost. At
the optimal concentration of SLO (0.15 U/mL), the platelet
population showed a normal distribution of sulforhodamine B
fluorescence, indicating that all platelets were
permeabilized (Figure 2B). This concentration
was used in subsequent experiments.
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Because permeabilization procedures are likely to affect cell function,
platelet aggregation and secretion were compared in intact and
SLO-treated platelets. Compared with intact platelets,
permeabilized platelets aggregated normally after
stimulation with -thrombin but slightly slower after stimulation
with ADP (Figure 3A).
[14C]Serotonin secretion was also
slower after permeabilization and amounted to 87±7% (n=7) of intact
platelets after 5 minutes of stimulation with 0.2 U/mL -thrombin
(Figure 3B). To assess whether permeabilized
platelets were capable of activating
IIbß3, the specific
binding of 125I-labeled fibronectin to
-thrombin–stimulated platelets was measured. SLO-treated
platelets showed 85±6% (n=8) of the binding of intact
platelets (Figure 3C). These data illustrate that despite a
slight decrease in aggregation, secretion, and ligand binding to
IIbß3,
permeabilized platelets preserved 
80% of their
functional responses.
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Although fibrinogen is the natural ligand for
IIbß3 in stirred
platelet suspensions, binding studies with -thrombin–stimulated
platelets are easily disturbed by fibrin formation. To confirm
previous findings that 125I-labeled fibronectin
and fibrinogen are bound to
IIbß3 with the same
kinetics,18 the specific binding of
125I-labeled fibrinogen to
IIbß3 was measured
under conditions that prevented -thrombin–induced fibrin formation.
Platelets were first incubated with -thrombin for 2 minutes and
then with the thrombin-neutralizing agent PPACK (30 nmol/L) for another
2 minutes. This was followed by 15 minutes in the presence of
125I-labeled ligand. As shown in Table 1, there was no significant difference
between the binding of fibronectin and fibrinogen. Also, the use of
PPACK did not interfere if the platelets were stimulated with an
optimal -thrombin concentration (0.2 U/mL). The presence of the
IIbß3
antagonist GRGDS abolished fibronectin and fibrinogen
binding by >95%, confirming that both ligands are bound to
IIbß3. A possible
binding of fibronectin to ß1 integrins on the
platelet was investigated in experiments with the
ß1 antagonist AIIB2. This antibody
changed neither fibronectin nor fibrinogen binding, indicating that the
binding data reflected primarily ligand binding to
IIbß3.
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To assess whether the activation state of integrin
IIbß3 was sensitive to
agents that do not penetrate intact cells, platelets were
stimulated with ADP (10 µmol/L) and -thrombin (0.2 U/mL),
which induced the binding of 22 537±6261 (n=4) and 59 045±9250
(n=8) fibronectin molecules per platelet, respectively (Figure 4). Although the cell-impermeable GDP
analogue GDPßS slightly reduced ligand binding to intact
platelets, the inhibition in permeabilized
platelets was much stronger, resulting in 80% (-thrombin) to
90% (ADP) inhibition by 400 µmol/L GDPßS. Together, these
results illustrate that permeabilized platelets
preserve sensitivity to inside-out regulation and at the same time are
accessible to membrane-impermeable agents.
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Because the permeabilization procedure was designed to introduce
peptides that possibly interfere with
IIbß3 regulation, a
peptide mimicking residues 749 to 756 in the ß3
tail, designated ß3-3, and a peptide mimicking
residues 755 to 7762, designated ß3-4 (Figure 1
), were labeled with FITC and added to the platelet
suspension before permeabilization. Subsequent FACS analysis
revealed a complete shift of the platelet suspension, indicating
that all platelets had accumulated a certain amount of the
FITC-labeled ß3-3 (Figure 5) and ß3-4 (not
shown). To further address the specificity of
IIbß3 inhibition by
the peptides, secretion studies were performed in the presence of
ß3-4 and in the presence of a peptide related
to an inhibitory peptide of Src kinases, named Src-R (see
below). None of these peptides interfered significantly with the
secretion of [14C]serotonin induced
by 0.2 U/mL -thrombin, illustrating that a major platelet
function was undisturbed (Table 2).
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Effect of Src and PKC Peptides on
IIbß3 Exposure
Earlier studies revealed that ligand binding to
IIbß3 was greatly
impaired in the presence of bisindolylmaleimide I and herbimycin A,
which are potent inhibitors of the PKC family and tyrosine
kinases, respectively. Because such a pharmacological approach might be
biased by aspecific side effects, the role of these protein kinases was
evaluated by introducing peptides that interfere with these enzyme
superfamilies. Permeabilized platelets stimulated
with -thrombin bound at 59 771±9775
125I-labeled fibronectin molecules per
platelet. To evaluate the role of Src kinases in affinity
regulation of IIbß3,
the Src peptide YIYGSFK was introduced. This is a substrate analogue of
Src kinases and competes with the natural substrate for
phosphorylation.23 The peptide
dose-dependently inhibited -thrombin–induced ligand binding, with
almost complete inhibition at 1000 µmol/L. In contrast, ligand
binding to intact platelets was unaffected (Figure 6A). The PKC inhibitory
peptide RKRCLRRL binds irreversibly to a conserved Cys residue in the
catalytic domain of PKC and inhibits the activity of PKC isoforms ,
ß, and in vitro.24 This peptide dose-dependently
reduced ligand binding, showing a maximal inhibition of 75%. No
effect of the PKC peptide was found in intact platelets. The
reversed peptide of the Src peptide with YF substitutions did not
change -thrombin–induced ligand binding (Src-R in Table 3) or
[14C]serotonin secretion (Table 2). To assess possible aspecific effects of these treatments,
peptides were introduced that were not related to signaling elements in
platelets. These were the peptides corresponding to domains of PCI,
named PCI-1 and PCI-2, and of ß2-gpI. None of these peptides
interfered significantly with -thrombin–induced ligand binding up
to a concentration of 1000 µmol/L (Table 3). Thus, these
data confirm the earlier findings based on pharmacological
inhibitors of PKC and tyrosine kinases and indicate more
specifically that Src kinases take part in the regulation of the
activation state of
IIbß3.
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To confirm that the peptides indeed interfered with their target
proteins, tyrosine-phosphorylated proteins (Figure 6B) and phosphorylated pleckstrin (Figure 6C) were analyzed. Unstimulated
permeabilized platelets showed tyrosine
phosphorylation of proteins with molecular masses of
50, 55, 60, 100, and 125 kDa, in accordance with earlier reports on
intact platelets.25 26 In addition, a weak tyrosine
phosphorylation was found in proteins of 84/87 kDa.
-Thrombin stimulation further increased the tyrosine
phosphorylation of these proteins and, in addition,
triggered phosphorylation of a protein with a molecular
mass of 70 kDa. The Src peptide inhibited these tyrosine
phosphorylations dose-dependently with almost complete
inhibition at 1000 µmol/L, which is in line with the expected
competition with the natural substrates (Figure 6D). In
contrast, neither the PKC peptide nor the reversed Src-R peptide
interfered with these tyrosine phosphorylations. As
expected, the PKC peptide strongly interfered with the
phosphorylation of pleckstrin, which in platelets
is a major PKC substrate. There was a dose-dependent reduction in
-thrombin–induced pleckstrin phosphorylation with a
maximum at 200 µmol/L. This phosphorylation
was unchanged in the presence of maximal concentrations (1000
µmol/L) of Src peptide and Src-R. Thus, the Src peptide and the PKC
peptide interfered with their respective target enzymes without showing
cross-reactivity. Therefore, their effect on the affinity regulation of
IIbß3 might result
from direct interference with the role of these kinases in inside-out
signaling to
IIbß3.
Identification of Cytoplasmic Domains in ß3 Involved
in Affinity Regulation of IIbß3
In Chinese hamster ovary cells, mutations and deletions in the
ß3 cytoplasmic tail interfere with the affinity
state of chimeric
IIbß3.4 11 27 28
These observations indicate that ß3 might be a
target for protein kinases that take part in inside-out signaling to
IIbß3, either via a
direct effect or via phosphorylation of 1 or more
proteins that control the affinity state of the integrin. To identify
these residues, 4 peptides were constructed that mimic different
domains of the ß3 cytoplasmic tail, as
illustrated in Figure 1. The peptide that resembled the membrane
proximal part of the ß3 tail, named
ß3-1, failed to interfere with the
-thrombin–induced fibronectin binding. In contrast, peptides
ß3-2, ß3-3, and
ß3-4 dose-dependently reduced
-thrombin–induced ligand binding (Figure 7A). At 500 µmol/L peptide,
ß3-2 reduced the binding by 60±4%,
ß3-3 reduced it by 97±1%, and
ß3-4 reduced it by 97±2% compared with ligand
binding to permeabilized platelets in the absence
of these peptides. The modified ß3-3 peptides
with an SA and an SP replacement showed the same inhibition as
ß3-3 (data not shown). No effect of these
peptides was seen on intact platelets (data not shown). These
results indicate that sequences at the carboxy-terminal end between
Asn743 and Thr762 of ß3 take part in the
regulation of the affinity state of
IIbß3.
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Because these peptides contain Ser, Thr, and Tyr residues and might
therefore be targets for protein kinases, a possible interference with
the natural substrates for PTKs and PKC was investigated by analyzing
-thrombin–induced tyrosine phosphorylation (Figure 7B) and pleckstrin phosphorylation (Figure 7C) in the presence of ß3-2,
ß3-3, and ß3-4.
Incubation with 500 µmol/L peptide did not change most of these
protein phosphorylations. An exception was the tyrosine
phosphorylation of the 20- and 100-kDa bands that
increased in the presence of the peptides (Figure 7D).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The major findings of the present study are as follows: (1)
-Thrombin initiates inside-out signaling to
IIbß3 via 1 or more
Src kinases and via PKC. (2) Residues 743 to 762 in the
carboxy-terminal end of the ß3 subunit contain
domains involved in the regulation of the activation state of
IIbß3. PTKs known to
be activated before ligand binding to
IIbß3 include Syk and
Src kinases.14 Because aggregation by -thrombin is
normal in Syk-deficient mouse platelets, this kinase is probably
not involved in inside-out signaling to
IIbß3.29
Platelets contain the Src kinases Src, Fyn, Yes, Lyn, and Hck, with
Src showing the greatest expression. Activation of Src follows
Gi-coupled receptor stimulation and is therefore
a very early step during platelet activation.30 The
YIYGSFK peptide is a specific substrate for all these Src kinases and
is phosphorylated at the N-terminus on Tyr-3 by Src,
Fyn, and Lyn with a Km of 55
µmol/L.23 It is a very poor substrate for membrane
receptor–linked PTKs and non–receptor-linked cytosolic PTKs other
than Src. The present results reveal complete inhibition of ligand
binding to IIbß3 at
1000 µmol/L YIYGSFK, indicating that 1 or more members of the
Src-family play a critical role in inside-out signaling to this
integrin. At present, it is uncertain which effectors are
controlled by Src. Because
IIbß3 is not
tyrosine-phosphorylated during the initial stages of
platelet activation and tyrosine phosphorylation of
the ß3-subunit critically depends on
aggregation,31 32 it is unlikely that the
ß3 subunit is a direct target of Src.
Alternatively, the inhibition of ligand binding by YIYGSFK might
reflect interference with the phosphorylation of an
integrin regulatory protein. A candidate is
ß3-endonexin, which contains a Tyr residue and
in Chinese hamster ovary cells binds to the ß3
cytoplasmic tail, thereby increasing ligand binding to
IIbß3.33
Another candidate is a calcium and integrin binding protein (molecular
mass 25 kDa), which contains 2 Tyr residues and binds to
IIb.34
Other potential substrates for Src kinases include Ras-GAP (120
kDa),35 the cytoskeleton protein cortactin (85/87 kDa),
and a number of unidentified proteins termed p54, p65, and p130 to
140.36 Src peptide inhibited tyrosine
phosphorylation of proteins with molecular masses of 55
and 100 kDa. Also, the tyrosine phosphorylation of a
60-kDa protein was reduced, possibly reflecting Src and interference
with the autophosphorylation of the positive regulatory
Tyr416 residue.14 Thus, each of these effector molecules
of Src is a candidate for a role in the affinity regulation of integrin
IIbß3. In addition to
activating PTKs, -thrombin is known to activate the Ser/Thr
kinase PKC. Platelets express the PKC isoforms , ß, 
, 
,
, and 
,37 38 39 which are translocated from the
cytosol to the membrane on platelet stimulation by -thrombin,
platelet-activating factor, or phorbol 12-myrisate
13-acetate.40 The RKRCLRRL peptide is an analogue of the
PKC peptide substrate RKRTLRRL, in which the Thr residue has been
substituted for a Cys residue. It inactivates the activity
of PKC, PKCß, and PKC by a covalent disulfide linkage to a
conserved site in the catalytic domain.24
-Thrombin–induced ligand binding to
IIbß3 was strongly
reduced by the PKC peptide, but the maximal inhibition was never
>75%, suggesting that part of the binding was insensitive to the
peptide. A similar, apparently PKC-insensitive, ligand binding is
observed in platelets treated with PKC inhibitors,
which amounts to 20%.12 These results indicate that
PKC contributes to
IIbß3 regulation, in
agreement with earlier work based on the PKC inhibitors
bisindolylmaleimide I and II and calphostin C in intact
platelets.12 Candidate substrates for PKC are the
ß3 tail of
IIbß316 41 42
and the regulatory protein ß3-endonexin, with 1 and
3 consensus motifs, respectively, for PKCs.
The ß3 cytoplasmic tail is a potential target
for intracellular signals generated by these kinases. The point
mutation Ser752Pro in the ß3 cytoplasmic
tail was associated with an apparent
IIbß3 activation
defect in a patient with Glanzmann’s
thrombasthenia.10 Prolonged inhibition of Ser/Thr
phosphatases by calyculin A increased the
phosphorylation of Thr753, which was accompanied by a
decrease in platelet adhesion and spreading on surface-coated
fibrinogen.42 In an attempt to compete with the native
integrin subunit for regulatory signals, peptides that mimic different,
but partially overlapping, domains of the ß3
subunit were designed. Peptide ß3-1, which
resembles the membrane-proximal part of the ß3
tail (residues 717 to 724), had no effect on -thrombin–induced
ligand binding. In contrast, peptides ß3-2
(residues 743 to 750), ß3-3 (residues 749 to
756), and ß3-4 (residues 755 to 762), which
mimic regions at the carboxy-terminal of the ß3
tail, interfered with agonist-induced ligand binding. Also, the
modified ß3-3 peptides with an SA (EATATFTN)
and an SP replacement (EATPTFTN) inhibited ligand binding,
indicating that the Ser residue (mimicking S752 in
ß3) is not critical for
ß3 function, in agreement with earlier findings
in HEL cells.43 No effect of these inhibitory
peptides was found on -thrombin–induced tyrosine
phosphorylation and phosphorylation of
pleckstrin, indicating that the interaction with the major substrates
of these kinases was left undisturbed.
The ß3 domains mimicked by these peptides
contain Tyr, Ser, and Thr residues. The Tyr residues in NNPLYKEA (743
to 750, mimicked by peptide ß3-2) and TNITYRGT
(755 to 762, peptide ß3-4) may be part of the
docking site of a regulatory protein. The NPXY motif (present in
peptide ß3-2) is highly conserved among many
cell surface receptors, including ß subunits of integrins, the LDL
receptor, and tyrosine kinase–linked receptors.41
Deletion of the NPXY motif or a Tyr747Ala substitution blocked
integrin function28 and abolished
IIbß3-mediated
internalization of fibrinogen-coated particles.44 Liu et
al43 found that the Tyr residues in the NPLY and NITY
motifs are essential in
IIbß3 affinity
regulation. A cell-permeable peptide mimicking residues 747 to 762 in
the cytoplasmic tail of
IIbß3 strongly reduced
adhesion of HEL and ECM304 cells to surface-coated
fibrinogen.44 Substitution of Tyr747 and/or Tyr759 for Phe
resulted in loss of the inhibitory function of the peptide.
The regulatory protein ß3-endonexin binds to
the NITY motif in the ß3 cytoplasmic
tail45 (residues 756 to 760), which might explain the
inhibitory effect of peptide ß3-4.
A second mechanism by which peptide ß3-3 and
ß3-4 may affect inside-out signaling is by
direct interference with the PKC-mediated
phosphorylation of the ß3
cytoplasmic tail. The ß3 tail is
phosphorylated on Thr after -thrombin
stimulation.46 A potential candidate is the Thr residue in
the NITY motif of ß3 (Thr758), which is in a
domain (TYR) that is recognized and
phosphorylated by PKC. Alternatively, Thr762 might also
be a target for PKC.47 In support of this concept are
findings in Chinese hamster ovary cells, in which deletion of the last
4 AAs (YRGT) of ß3 disrupted the TYR motif and
blocked binding of the antibody PAC-1.27
The present results in combination with reported data support the
concept that activation of Src kinases is a common step in inside-out
signaling to IIbß3 by
most platelet-activating agents. In addition, PKC contributes to
inside-out signaling in platelets stimulated by -thrombin.
Inhibition of ligand binding to
IIbß3 by peptides
mimicking the carboxy-terminal part of the ß3
tail shows that residues 749 to 762 are likely targets for signals
generated by these kinases.
|
Acknowledgments |
|---|
This study was supported in part by a grant from the Netherlands Thrombosis Foundation (No. 92002) and a grant from the Netherlands Organization for Scientific Research/Netherlands Heart Foundation (No. 902-526-094/940-50-102). G.v.W. is a research fellow of the Catharijne Foundation.
Received July 23, 1999; accepted November 25, 1999.
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References |
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