© 1999 American Heart Association, Inc.
Thrombosis |
Activation of Protein Kinase C Is Required for the Stable Attachment of Adherent Platelets to Collagen but Is Not Needed for the Initial Rapid Adhesion Under Flow Conditions
From the Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville.
Correspondence to Dr Renata Polanowska-Grabowska, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908. E-mail rp4t{at}virginia.edu
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Abstract—We have investigated the role of protein kinase C (PKC) in the initial events of
2ß1-integrin–mediated platelet
adhesion to collagen under flow conditions. Although adhesion caused
activation of PKC, as evidenced by pleckstrin
phosphorylation, the PKC inhibitors GF
109203X and Gö 6976 had no effect on adhesion, even though they
prevented pleckstrin phosphorylation. The initial
kinetics and extent of platelet adhesion to collagen (<5 seconds)
and tyrosine phosphorylation of p125FAK and
p72syk were not influenced by the PKC
inhibitors, whereas adhesion to polylysine was prevented.
These results indicate that adhesion to collagen and polylysine involve
different mechanisms and requirements for PKC activation. Pretreatment
with GF 109203X destabilized collagen-adherent platelets,
accelerating their detachment, which was associated with tyrosine
dephosphorylation of p125FAK. Thus,
although PKC activation was not required for rapid platelet
adhesion to collagen, it appears to play an important role in
stabilizing the attachment of adherent platelets to collagen. We
also examined the effect of PKC activation by the phorbol ester phorbol
12-myristate 13-acetate (PMA) on platelet adhesion to
collagen. PMA at 100 nmol/L strongly potentiated adhesion and tyrosine
phosphorylation of p125FAK and
p72syk and activated ß1-integrins, as
determined by increased exposure of the 15/7 epitope. The
PMA-stimulated adhesion was partially blocked by an
anti-2ß1 antibody, was completely
inhibited by GF 109203X, and was not correlated with the extent of
pleckstrin phosphorylation. Therefore, strong PKC
activation may lead to inside-out signaling, enhancing the role of
ß1-integrins in adhesion. Pleckstrin
phosphorylation does not appear to be involved in the
initial phase of basic or PMA-stimulated adhesion but may help
stabilize the adherent platelets.
Key Words: platelets • adhesion • collagen • PKC • p125FAK • p72syk
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Introduction |
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Protein kinase C (PKC) was initially identified as a threonine/serine protein kinase dependent on calcium and phospholipids and known to be activated during the earliest events of signal transduction, tumor promotion, and cell regulation.1 2 3 4 Activation of PKC has been linked to reorganization of the actin cytoskeleton, cell motility, adhesion, and the formation of focal contacts.5 6 7 The involvement of PKC in cell adhesion to extracellular matrix (ECM) proteins has long been proposed from the observations that direct stimulation of PKC by phorbol esters can activate integrins, potentiating adhesion and spreading, and that inhibitors of PKC prevent adhesion.8 9 10 11 12 13 There is also evidence that direct integrin binding to ECM proteins leads to PKC activation.9 14 Although stimulation of PKC by phorbol esters can mediate integrin activation and enhance adhesion, there is little evidence that PKC directly regulates these events.
Platelets are a good model to study signal transduction events during attachment of cells to ECM proteins or for cell-cell interactions.15 Several studies suggest that PKC plays an important role in blood platelet activation associated with granule secretion and aggregation.16 17 18 In particular, prior activation of PKC with PMA desensitizes platelets to thrombin stimulation, as indicated by an accelerated inositol trisphosphate metabolism and decreased internal Ca2+ mobilization.19 Much interest has focused on a protein of apparent molecular mass of 47 kDa, called pleckstrin, which is phosphorylated when platelets are activated by agonists that directly or indirectly activate PKC.20 Phosphorylation of pleckstrin correlates closely with serotonin secretion from platelet-dense granules and is frequently used as a marker of PKC activation in human platelets.21 22 More recently, it was shown that pleckstrin phosphorylation in platelets exposed to thrombin or to the thrombin receptor–activating peptide (TRAP) is also partially dependent on activation of phosphoinositide (PI) 3-kinase.23 24
In platelets, as in other cell types, adhesion to collagen causes tyrosine phosphorylation of a 125-kDa cytosolic tyrosine kinase, known as a focal adhesion kinase p125FAK, a process strongly correlated with the formation of focal contacts.25 26 Signaling molecules that have been shown to be activated during collagen stimulation include the p72syk tyrosine kinase.27 The initial phase of protein tyrosine phosphorylation of several tyrosine kinases depends on prior PKC activation,28 and it has been proposed that protein tyrosine phosphorylation in platelets occurs after activation of PKC.29 This suggests that PKC activation may also influence the initial stage of tyrosine phosphorylation of p125FAK and p72syk after platelet adhesion. Tyrosine phosphorylation of p125FAK in platelets stimulated with a mixture of epinephrine and anti-LIBS6 antibody30 or in platelets adherent either to fibrinogen or to immobilized immunoglobulin IgG31 32 is blocked when PKC activity was inhibited with a specific PKC inhibitor, bisindolylmaleimide GF 109203X, suggesting that p125FAK is activated downstream of PKC. PKC-induced serine phosphorylation of p125FAK has been suggested to regulate the intracellular stability of focal adhesion kinase in mouse 3T3 cells.33
Platelet adhesion to an exposed subendothelium is a
critical physiological process that must occur
rapidly at the site of vessel-wall injury.34 Previously,
we have studied the mechanisms of platelet adhesion to collagen
under flow conditions, with shear parameters similar to
those in microcirculation.35 Platelet adhesion under
these conditions is very efficient, occurring within seconds, and in a
plasma-free, Mg2+-containing buffer it is
mediated primarily by the
2ß1-integrin,36
although other receptors or associated proteins may
participate.37 38 39 40
In the present study, we investigated the involvement of PKC in platelet adhesion to collagen under flow conditions by using several PKC inhibitors. In cases of vessel-wall injury, not only must platelet adhesion to the exposed adhesive proteins of the ECM be rapid (seconds to minutes), but also, the stability of the attached platelets must be sufficient to prevent blood loss and contribute to the healing process. Our continuous-flow approach36 for following adhesion kinetics as well as biochemical changes provides an opportunity to study separately the roles of PKC during the initial attachment of platelets to collagen and in stabilizing the collagen-platelet interaction. Because adhesion can activate PKC and cause tyrosine phosphorylation of p125FAK and p72syk and because these kinases are present in focal contacts,41 42 43 we also examined the influence of PKC activation by a phorbol ester on adhesion and the tyrosine phosphorylation of these kinases.
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Materials
Type I collagen from rat skin was a gift from Dr G. Balian (University of Virginia, Charlottesville). Polylysine, BSA, indomethacin, prostaglandin I2 (PGI2), apyrase (grade VII), phorbol 12-myristate 13-acetate (PMA), and bovine thrombin were from Sigma Chemical Co. The PKC inhibitors indolocarbazole Gö 6976 and bisindolylmaleimide I (GF 109203X) were from Calbiochem-Novabiochem Corp. [32P]orthophosphoric acid was from DuPont NEN. A monoclonal antibody (mAb) 4D10 against p72syk was purchased from Santa Cruz Biotechnology, Inc. The mAbs 2A7 and BC3 against p125FAK were gifts from Dr T. Parsons (University of Virginia, Charlottesville). The mAbs 4G10 and PY20 against phosphotyrosine were purchased from Upstate Biotechnology Inc and Transduction Laboratories, respectively. An mAb 15/7 that recognizes a high-affinity or ligand-occupied conformation of ß1-integrin44 was a gift from Dr T. Yednock (Athena Neurosciences Inc). FITC-conjugated goat anti-mouse IgG1 and purified mouse IgG1 were from Zymed Laboratories Inc. Anti–human integrin-
2 and anti–human
integrin-5 mAbs, P1E6 and P1D6, respectively,
were from Gibco/BRL. An anti–ß1-integrin mAb
1959 was from Chemicon. GRGDSP peptide was from Peninsula Laboratories.
The alkaline phosphatase–conjugated goat anti-mouse secondary antibody
and the chemiluminescence-based Western blot detection system were
from Bio-Rad.
Platelet Preparation
After informed consent had been obtained, platelet-rich
plasma was obtained from fresh human blood anticoagulated with
acid-citrate-dextrose (ACD: 120 mmol/L sodium citrate, 110
mmol/L glucose, 80 mmol/L citric acid) by
centrifugation as described.45
Platelets were isolated from platelet-rich plasma by
centrifugation at 620g for 20 minutes in the
presence of 0.05 volumes of ACD, apyrase (7.5 U/mL ADPase activity),
indomethacin (1 µg/mL), and
PGI2 (0.3 µg/mL). For labeling,
1010 platelets were resuspended in 9 mL of
phosphate-free buffer (140 mmol/L NaCl, 5 mmol/L KCl,
0.05 mmol/L CaCl2, 0.1 mmol/L
MgCl2, 0.01 g/mL BSA, 16.5 mmol/L glucose,
15 mmol/L HEPES, pH 7.4) containing 1 µg/mL
indomethacin and 7.5 U/mL apyrase. The platelet
suspension was left for 20 minutes at room temperature, and the
labeling process was performed by incubating platelets with 1 mCi
carrier-free [32P]orthophosphate for 90 minutes
at 37°C. Subsequently, platelets were centrifuged, washed
again, and resuspended at a concentration of
4x108 to 6x108/mL in a
modified Tyrode’s buffer (140 mmol/L NaCl, 0.34 mmol/L
Na2HPO4, 2.9 mmol/L
KCl, 10 mmol/L HEPES, 12 mmol/L NaHCO3,
5 mmol/L glucose, pH 7.4) containing 2 mmol/L
MgCl2. In selected experiments,
inhibitors of platelet activation apyrase (7.5 U/mL),
indomethacin (1 µg/mL), and GRGDSP peptide (500
µmol/L) were included before adhesion assays were performed.
Platelet suspensions were usually prewarmed at 37°C for 10
minutes before addition of compounds as appropriate.
Platelet Adhesion Assay
The continuous-flow adhesion approach was essentially as
described previously.35 BrCN-activated Sepharose
beads are coated with native soluble collagen type I from rat skin or
in control experiments with polylysine or BSA and used as adhesive
substrates. One syringe containing washed platelets and a second
syringe containing isotonic saline are connected via Teflon tubing to
the microadhesion column. Their contents are mixed and pumped through
the protein-coated beads. The flow rates through the beads are
regulated by a variable syringe pump and give shear rates at the
bead surface from 850 to 3400 s-1. Adhesion to
collagen is determined by counting of single platelets in the
suspension before and after exposure to the beads with a resistive
particle counter and is expressed as the percentage of platelets
bound to collagen. To determine the changes in protein
phosphorylation, we usually used platelets that had
adhered to 50 µL of protein-coated beads at a pumping speed of 3.4
µL/s, which yields a shear rate of 1700 s-1.
Platelets (250 µL; 1x108 to
1.5x108) were pumped at this shear rate for
90 seconds. To obtain sufficient amount of proteins for
immunoprecipitations (1 to 2 mg), we used 109
platelets per immunoprecipitation. PMA and PKC
inhibitors were prepared in 0.1% (vol/vol) DMSO. When the
effects of PKC inhibitors were studied, platelets were
pretreated with these inhibitors or with 0.1% DMSO to
serve as controls for 10 minutes at 37°C. In some experiments,
adhesion studies were performed after an initial preincubation for 10
minutes with apyrase (7.5 U/mL) and indomethacin (1
µg/mL), followed by 3 minutes of treatment with 20 or 100 nmol/L PMA
without stirring. To investigate the PMA-stimulated adhesion to
collagen, we used beads coated with half the amount of collagen
compared with the original beads used for our other
experiments.35
Platelet Detachment Assay
Washed platelets preincubated either with the PKC
inhibitor GF 109203X (12 µmol/L) or with 0.1% DMSO
(control) were pumped through the adhesion column for 90 seconds as
described above. Two different approaches were used to study
detachment. In the first, beads with adherent platelets were
immediately washed with saline, suspended in a
Mg2+-containing medium, and shaken for 10, 30,
60, 120, and 240 minutes at 37°C in a shaking water bath at 60
strokes/min and with a stroke length of 30 mm. In the second
approach, platelets adherent to the collagen-coated beads were left
in the column for 10, 30, 60, 120, and 240 minutes in the presence of
physiological buffer, and subsequently, buffer was
pumped through the column for 6 minutes under constant shear. In both
approaches, detachment of platelets was determined by counting of
the released platelets in the suspending buffer or in the effluent
from the column, respectively. The phosphorylation of
p125FAK was determined at the same time points.
Lysis buffer was added to every tube containing collagen beads with
platelets and buffer. Platelet proteins were concentrated and
then used for immunoprecipitation with
anti-p125FAK mAb BC3, followed by Western
blotting with anti-phosphotyrosine and
anti-p125FAK antibodies. The protein bands were
visualized by chemiluminescence. The phosphorylation of
p125FAK determined by densitometry at defined
time points is expressed as a percentage of the tyrosine
phosphorylation measured at time 0, when all
platelets were attached to the beads.
Gel Electrophoresis and Western Blotting
The same amounts of adherent, aggregated, and control
platelets were lysed in SDS-containing buffer (2% SDS [wt/vol],
5% 2-mercaptoethanol [vol/vol], 10% glycerol [vol/vol], 0.002%
bromphenol blue [vol/vol], and 62.5 mmol/L Tris, pH 6.8) and
heated for 5 minutes at 95°C. Platelet lysates (from
1.1x107/lane) were separated by 8% or 12%
SDS-PAGE and electrotransferred from the gels onto nitrocellulose
membranes. To block residual protein-binding sites, membranes were
incubated overnight with 5% BSA (vol/wt) in TBS-T buffer (150
mmol/L NaCl, 50 mmol/L Tris [pH 7.5], and 0.1% [vol/vol]
Tween 20). The blots were then washed and incubated for 2 hours with
anti-p125FAK, anti-p72syk,
or anti-phosphotyrosine antibodies. Membranes were washed 4 times in
TBS-T, and bound primary antibody was detected by incubation with
alkaline phosphatase–conjugated goat anti-mouse IgG as a secondary
antibody diluted 1:3000. Membranes were washed and treated with ECL
reagents before exposure to ECL-Hyperfilm (Amersham).
Immunoprecipitations
Adherent platelets (5x108/500 µL)
were lysed in an ice-cold lysis buffer as described.25
Insoluble cell debris was removed by centrifugation at
3000g for 5 minutes. The supernatant was precleared with
protein A-Sepharose CL-4B, removed, and incubated for 18 hours at 4°C
with 5 µg of anti-p125FAK (2A7), with 5 µg of
anti-p72syk (4D10), or with 5 µg of an
appropriate control antibody. Anti-mouse IgG–adsorbed protein A beads
were then added, and the immune complexes were washed twice with lysis
buffer and twice with ice-cold PBS buffer, pH 7.2, before resuspension
in SDS sample buffer. Immunoprecipitated proteins were subjected to 8%
SDS-PAGE, transferred to nitrocellulose, and probed as described
above.
Fluorescence-Activated Cell Sorting
Analysis of Platelets
Washed platelets were resuspended in a modified Tyrode’s
buffer containing 0.1% BSA, 2 mmol/L Mg2+,
10 µmol/L indomethacin, and 7.5 U/mL apyrase at
108/mL and were incubated with PMA (20 or 100
nmol/L) or with 0.1% DMSO for 3 minutes without stirring at 37°C.
The platelet stimulations were stopped by the addition of 100
volumes of PBS buffer, pH 7.2, containing aggregation
inhibitors and centrifugation. Then the
platelets were resuspended in a Tyrode’s buffer, followed by the
addition of the appropriate mAb, such as 15/7, P1E6, 1959, or control
mouse IgG1. Subsequently, anti-mouse FITC-conjugated antibody was
added, and after 30 minutes, the platelets were fixed by the
addition of an equal volume of 2% (wt/vol)
paraformaldehyde in PBS buffer, pH 7.2, followed by
centrifugation. Finally, platelets were washed and
resuspended in Tyrode’s buffer at 5x106/mL and
analyzed for FITC fluorescence on a Becton-Dickinson
FACStar. In some experiments, before stimulation with PMA,
platelets were pretreated with the PKC inhibitor GF
109203X (12 µmol/L) for 10 minutes.
Statistical Analysis
When appropriate, evidence for significant differences between
control and test situations was assessed by Student’s t
test for paired samples. Data are usually presented as
mean±SD.
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Results |
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Pleckstrin Phosphorylation After Platelet Adhesion to Collagen
One-dimensional gel electrophoresis of 32P-labeled human platelets showed that platelet adhesion to collagen under flow conditions stimulated a marked increase in pleckstrin phosphorylation of 3.2±0.8-fold within 90 seconds of perfusion (Figure 1A
). Under the same flow conditions,
phosphorylation of pleckstrin also occurred in
platelets adherent to the nonspecific substrate polylysine, albeit
to a greater extent (6.5±0.5-fold) than that observed in platelets
adherent to collagen (Figure 1A). As expected, platelets
stimulated either with the G protein–coupled receptor agonist thrombin
(1 U/mL) or by a direct activator of PKC, PMA (100 nmol/L),
also revealed strong pleckstrin phosphorylation
(7.0±0.7- and 6.7±0.8-fold, respectively). Analysis of
platelets in the effluent from the microcolumn showed that neither
shear forces acting alone nor possible brief platelet-collagen
contact during flow through the beads caused pleckstrin
phosphorylation (Figure 1A). Nonspecific
adhesion to BSA was <5% and did not result in pleckstrin
phosphorylation (data not shown). Because the
stimulation of pleckstrin phosphorylation might be
enhanced by secondary events, such as formation of
arachidonate metabolites, release of ADP, or
IIbß3-dependent
aggregation, we also evaluated pleckstrin
phosphorylation in adherent platelets in the
presence of the cyclooxygenase
inhibitor indomethacin apyrase, which
removes ADP that might be inadvertently released from
the platelets, and the GRGDSP peptide, which blocks fibrinogen
binding and aggregation. None of these inhibitors used
alone or in combination influenced pleckstrin
phosphorylation, indicating that adhesion to collagen
activated PKC independently of these factors (Figure 1B).
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PKC Inhibitors Do Not Influence Rapid Platelet
Adhesion to Collagen: Effects on Pleckstrin Phosphorylation
To investigate the role of PKC in platelet adhesion, we
examined whether a prior exposure to PKC-specific
inhibitors modified the adhesion kinetics and pleckstrin
phosphorylation. We tested adhesion in the presence of
2 frequently used specific PKC inhibitors: the
indolocarbazole Gö 6976, which preferentially inhibits
Ca2+-dependent isozymes46 and
bisindolylmaleimide GF 109203X, which interacts with the catalytic
subunit of PKC and blocks both Ca2+-dependent and
Ca2+-independent isozymes.47 We
found that pretreatment of platelets with 0.5 to 20 µmol/L
GF 109203X did not influence efficiency of adhesion to collagen (within
90 seconds of perfusion) (Figure 2A).
Under the same conditions, adhesion to polylysine was reduced in a
dose-dependent manner, reaching a maximal level of inhibition (85%) at
10 µmol/L GF 109203X (Figure 2A). Similar behavior was
found for pretreatment with Gö 6976 at 1 to 15 µmol/L;
adhesion to collagen was not affected, whereas inhibition of adhesion
to polylysine reached maximum (80%) at 10 µmol/L. In most
subsequent studies, we used single concentrations of
inhibitors, 12 µmol/L GF 109203X and 10
µmol/L Gö 6976, which are known to inhibit PKC
activity46 47 and have often been used in the literature.
Pretreatment with these inhibitor concentrations did not
affect the initial kinetics (<5 seconds) of platelet adhesion to
collagen (Figure 2B), whereas adhesion to polylysine was almost
completely blocked. Preincubation with GF 109203X in the range of 0.5
to 20 µmol/L inhibited pleckstrin
phosphorylation in collagen-adherent platelets in a
dose-dependent manner, reaching almost complete inhibition at 5
µmol/L (data not shown). At 12 µmol/L, GF 109203X inhibition
was 98±7%, and in polylysine-adherent platelets it was 95±8%
(data not shown). Gö 6976 at 10 µmol/L partially blocked
pleckstrin phosphorylation induced by adhesion to
collagen (65±4%, Figure 3), whereas it
strongly blocked phosphorylation caused by polylysine
(89±6%, data not shown).
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PKC Inhibition Does Not Influence Adhesion-Induced Tyrosine
Phosphorylation of p125FAK and
p72syk
Because GF 109203X was the most potent and specific
inhibitor of PKC and it effectively blocked pleckstrin
phosphorylation in collagen-adherent platelets, we
chose this compound to study tyrosine phosphorylation
of p125FAK and p72syk. We
previously showed that rapid adhesion to collagen but not to polylysine
under flow conditions causes tyrosine phosphorylation
of p125FAK.25 In the present
study under similar flow conditions, p72syk was
also tyrosine phosphorylated in collagen-adherent
platelets (Figure 4). Platelet
preincubation either with DMSO or with GF 109203X had no effect on
overall protein tyrosine phosphorylation in either
control or adherent cells (data not shown). As illustrated in Figure 4, GF 109203X did not influence tyrosine
phosphorylation of p125FAK
(Figure 4A) and p72syk (Figure 4B)
induced by platelet adhesion to collagen. Western blotting revealed
similar protein levels of p125FAK and
p72syk before and after platelet exposure to
the inhibitor.
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PKC Inhibitors Stimulate Platelet Detachment
From Collagen
To investigate a possible role of PKC in maintaining stable
attachment of platelets to collagen, we studied the effect of GF
109203X on spontaneous detachment of adherent platelets from the
collagen surface. Platelets preincubated with the PKC
inhibitor that had adhered to the collagen-coated beads
revealed enhanced detachment from the collagen-coated beads compared
with DMSO-treated controls both after 6 minutes of flushing of the
column at a shear rate of 850 s-1 (Figure 5A) and after time spent in a shaking
bath (Figure 5B). Detachment was correlated with decreased
tyrosine phosphorylation of
p125FAK (Figure 5C), and it was not
associated with changes in FAK amount, as determined by an
immunoblot using anti-p125FAK
antibody (data not shown). These results suggest involvement of PKC in
maintaining platelet attachment after the initial binding to
collagen.
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Effects of PMA Stimulation
Several reports indicate that phorbol esters enhance adhesion of
different cell types to adhesive proteins without having any effect on
the adhesion to nonspecific substrates.48 49 50 In addition,
phorbol esters may desensitize receptor-mediated responses, such as
thrombin-stimulated internal Ca2+ mobilization
and formation of inositol phosphates.19 In the present
study, platelets in the presence of indomethacin
and apyrase were preincubated with 20 nmol/L PMA for 3 minutes without
stirring and used for adhesion. Treatment with this low dose of PMA
caused strong phosphorylation of pleckstrin (Figure 6A) and a weak tyrosine
phosphorylation of p72syk (Figure 6B) and p125FAK (Figure 6C),
without having any effect on adhesion (Figure 7). PMA at 100 nmol/L and 1 µmol/L
(data not shown) caused an increase in phosphorylation
of pleckstrin similar to that observed for 20 nmol/L (Figure 6A). However, tyrosine phosphorylation of
p125FAK and p72syk was much
stronger at 100 nmol/L PMA than at the lower dose of PMA (Figure 6B and 6C). Adhesion was significantly accelerated and
potentiated by this PMA treatment (Figure 7). Preincubation with
GF 109203X reversed the effect of PMA on the extra level of adhesion,
bringing it down to control levels (Figure 7). To determine
whether the stimulatory effects of PMA would result from PMA-induced
aggregation, PMA-dependent adhesion was studied in the presence of
500 µmol/L GRGDSP peptide. The RGD-containing peptide had no
effect on PMA-stimulated adhesion to collagen. We also examined whether
PMA could stimulate platelet adhesion to the nonspecific substrate
BSA and did not observe any significant adhesion to BSA-coated
beads.
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Inhibition of PMA-Stimulated Adhesion to Collagen With
Anti-2ß1 Antibody
We have previously shown that the antibody 6F1 against the
2ß1-integrin inhibits
up to 80% platelet adhesion to collagen in the presence of
Mg2+ in a plasma-free buffer.25 36
In contrast, 6F1 antibody did not prevent platelet adhesion to
polylysine (data not shown). In the presence of the 6F1 antibody,
PMA-stimulated adhesion (after 90 seconds of perfusion at a contact
time of 1.35 seconds) dropped from 70±4% to 21±3%. In comparison,
"basic" adhesion of untreated platelets dropped from 36±5% to
7±2% under the same conditions. Therefore, the extra adhesion due to
PMA stimulation was blocked by 60% by the
anti-2ß1 antibody. In
contrast, PMA-dependent adhesion was not blocked by an antibody (P1D6)
against the
5ß1-integrin.
PMA Stimulation May Cause a Conformational Change in
ß1-Integrins
Because PMA-stimulated adhesion to collagen was partially
inhibited by the
anti-2ß1 antibody 6F1
and because PMA may stimulate inside-out signaling events, resulting in
activation of integrins,51 we investigated whether PMA
directly affects platelet ß1-integrins. We
used the monoclonal antibody 15/7, which recognizes an
activation-dependent epitope on the ß1-subunit
of integrins.44 Fluorescence-activated
cell sorting (FACS) analysis showed that treatment with 100
nmol/L PMA (but not with 20 nmol/L PMA or with DMSO) markedly increased
(up to 30%) the binding of the 15/7 antibody to platelets. In
contrast, platelet stimulation with 100 nmol/L PMA did not increase
the binding of the control antibody, purified mouse myeloma IgG1, or
anti-ß1 mAb 1959. Similarly, the binding of the
anti-2 mAb P1E6 was unchanged by this
treatment. The increased binding of the 15/7 antibody to platelets
was inhibited by pretreatment with GF 109203X.
Downregulation of PKC by PMA
Because prolonged treatment of cells with PMA downregulates
PKC,4 we examined how this treatment influences
platelet adhesion to collagen. Platelets treated with the
combination of the GRGDSP peptide, indomethacin, and
apyrase were incubated without stirring with 100 nmol/L PMA or DMSO
(control) for 3 and 30 minutes and 2, 4, and 6 hours at 37°C.
DMSO-treated platelets exhibited only basic adhesion, identical to
untreated platelets. Preincubation with PMA for up to 2 hours
potentiated platelet adhesion to the same degree (30% to 40%)
as preincubation for shorter times (3 to 30 minutes). Protein
phosphorylation patterns, specifically pleckstrin
phosphorylation, were not altered during 2 hours of
incubation compared with 3- and 30-minute incubations (Figure 8). However, pretreatment with PMA for 4
hours no longer potentiated adhesion and did not influence initial
adhesion kinetics, even though it caused higher
phosphorylation of pleckstrin in DMSO-treated controls.
A 6-hour incubation with PMA caused an additional decrease in
pleckstrin phosphorylation compared with 4-hour
treatment, and the initial adhesion rate was the same as in basic
adhesion (DMSO-treated controls or untreated platelets). The extent
of adhesion after this treatment was significantly decreased (up to
50%) compared with control platelets when perfusion times were
lengthened to 90 seconds. In addition, the platelet detachment
assay using the shaking bath showed that the platelets that had
adhered to collagen beads after 6-hour treatment with PMA were also
rapidly detached from collagen, with an initial rate (5.5%/min) that
was faster than that observed for GF 109203X–treated platelets
(3.5%/min) or controls (1%/min). Almost complete detachment of
PMA-treated platelets from collagen-coated beads was observed after
1 hour of shaking. Protein levels visualized with Coomassie stain did
not indicate any proteolytic degradation up to 6 hours, and the amount
of p125FAK determined by Western blotting with
anti-p125FAK antibody was constant (data not
shown).
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Platelet Adhesion to Collagen: Activation of PKC and Effects of PKC Inhibitors
We have shown that platelet adhesion to collagen under flow conditions induced rapid PKC activation, evidenced by pleckstrin phosphorylation (Figure 1
). Strong
phosphorylation of pleckstrin also occurred during
adhesion to positively charged polylysine, frequently regarded as a
nonspecific control substrate. The phosphorylation of
pleckstrin in collagen-adherent platelets was significantly less
than in polylysine-, thrombin-, or PMA-stimulated platelets,
suggesting a lower degree of PKC activation or involvement of different
PKC isozymes. Pleckstrin phosphorylation after
platelet stimulation by collagen fibrils has been observed in
earlier studies.52 53 54 55 56 57 However, those studies involved
collagen-induced aggregation and secretion associated with secondary
activation, and only a small proportion of the platelets actually
adhered to the collagen fibrils. In our system, the
2ß1-mediated rapid
adhesion to collagen under arterial-flow conditions was not
associated with significant secretion of serotonin from
dense granules or formation of platelet aggregates.35
Therefore, the PKC activation we observed occurred as a direct result
of platelet binding to collagen and before secondary events, such
as aggregation and secretion.
PKC-selective inhibitors had varied effects on pleckstrin
phosphorylation in collagen- and polylysine-adherent
platelets. The bisindolylmaleimide GF 109203X, which inhibits both
Ca2+-dependent and
Ca2+-independent PKC isozymes,47
almost completely prevented phosphorylation of
pleckstrin in platelets adherent to collagen and to polylysine. In
contrast, Gö 6976, which is known to block
Ca2+-dependent PKC isozymes,46
inhibited the pleckstrin phosphorylation in
polylysine-adherent cells up to 90% but caused only partial (65%)
inhibition in collagen-adherent platelets (Figure 3). The
differences in inhibition of pleckstrin phosphorylation
in collagen- and polylysine-adherent platelets suggest that
Ca2+-independent PKC isozymes are
activated in collagen-specific adhesion but not in adhesion to
polylysine.
When PKC activity was blocked by GF 109203X or Gö 6976, the
initial kinetics and extent of platelet adhesion to collagen with
90 seconds of perfusion were unchanged (Figure 2), whereas
adhesion to polylysine was strongly (up to 85%) inhibited. This
suggests that PKC activation is required for platelet attachment to
polylysine but not for
2ß1-integrin–mediated
adhesion to collagen and that PKC activation does not regulate the
initial attachment to collagen.
Because PKC activation occurred rapidly after platelet binding to
collagen, we investigated whether it may play a role in later stages of
adhesion. We found that platelets treated with the PKC
inhibitor GF 109203X were detached from the collagen
surface much faster than untreated controls over extended time periods
(from minutes up to 4 hours) after the initial platelet-collagen
interaction (Figures 5A and 5B). The first version of the
detachment assay (using 6 minutes of flushing with
physiological buffer) yielded 3-fold greater
platelet detachment (Figure 5A). This method was less harsh
than the shaking bath (Figure 5B)—after 1 hour, 10%
detachment occurred in the control compared with 45% due to
shaking—and may better simulate conditions in the microcirculation.
Detachment caused by the more gentle flushing conditions did not
significantly increase with time, in contrast to the results from the
shaking bath, in which the effects of continuous mechanical stress
accumulated over time. The accelerated platelet detachment was
associated with decreased tyrosine phosphorylation of
p125FAK (Figure 5C).
The prolonged exposure of platelets to PMA (4 and 6 hours), which downregulates PKC, had no effect on initial adhesion rates (<1 second) and is consistent with our findings with PKC inhibitors. This treatment caused accelerated detachment of adherent platelets similar to that observed for GF 109203X–treated platelets. Interestingly, a 6-hour treatment with PMA also caused a partial (up to 50%) decrease in adhesion efficiency within 90 seconds of perfusion, compared with the 4-hour PMA treatment (which yielded the same adhesion as untreated controls). This is most likely due to higher detachment rates of initially adherent platelets during 90 seconds of perfusion, in contrast to the 4-hour PMA treatment. It appears that a significant portion of platelets pretreated with PMA for 6 hours, which initially adhere to collagen, are only weakly attached and are unable to resist shear forces and maintain stable attachment under flow for 90 seconds.
Our results suggest that PKC activation directly or indirectly plays an
important role in maintaining platelet attachment to collagen and
that adhesion-induced PKC activation, although not required in the
initial phase of adhesion, helps stabilize the platelet-collagen
interaction. This effect could well be important in hemostasis for
maintaining effective platelet adherence to exposed adhesive
proteins at sites of vessel-wall injury. Possibly, a sustained PKC
stimulation downstream of the
2ß1-integrin binding
to collagen may provide positive feedback required to maintain the
receptor in an active conformation. PKC may also play a role in the
stabilizing function of other platelet collagen
receptors.37 38 39 40
Although there have been no studies of PKC involvement in platelet adhesion under flow conditions, several reports indicate that PKC inhibitors block adhesion under static conditions. In particular, Vuori and Ruoslahti9 reported that calphostin C inhibits fibroblast adhesion to fibronectin. PKC inhibitors also block platelet adhesion to von Willebrand factor and immunoglobulins.32 58 At first sight, these observations might seem to contradict our results. In these studies, however, adhesion is performed under static conditions and over time scales longer than half an hour. Under such conditions, not only would the initial cell attachment be observed, but also, the subsequent detachment could be accelerated by PKC inhibitors, leading to poorer retention of the initially adherent cells. The fact that adhesion in our system was not markedly blocked by PKC inhibitors could also be explained by a rapid adhesion under flow conditions, causing only limited activation of PKC, seen as a relatively low phosphorylation of pleckstrin and lack of secretion and aggregation, which are known to be closely associated with PKC activation. In contrast, most of the traditional platelet adhesion assays discussed in the literature involve a degree of platelet aggregation and secretion. In such assays, a combination of platelet adhesion, secretion, and aggregation might strongly activate PKC and is likely to be more sensitive to PKC inhibitors. This could help explain the difference between our results and the other findings32 58 and may reflect different situations in vivo: monolayer adhesion to vessel walls in the microcirculation (our system35 ) and multilayer adhesion/aggregation occurring during thrombus formation when methods used are based on the Baumgartner approach.34
There are several other assay systems for studying platelet adhesion under flow, and although adhesion characteristics can be followed, they do not provide the ready ability to carry out biochemical analyses on the adherent platelets, which is an important aspect of our system.25 35 36 Sixma et al59 recently described a microperfusion chamber based on earlier designs of this group, which was used for following adhesion kinetics at shear rates of 1600 s-1. These conditions are very similar to our flow-through adhesion column containing collagen-coated Sepharose beads.35 "Stagnation-point" flow systems represent yet another approach60 61 and involve directing a jet of cells onto a surface where adhesion can be monitored. However, as indicated above, biochemical studies of the adherent platelets are not feasible.
PKC activation has been linked to tyrosine
phosphorylation of
p125FAK.15 There are several
distinct types of receptors that might signal tyrosine
phosphorylation of p125FAK and
p72syk. These include integrins,62
Fc
receptors,63 various bioactive lipids such as
lysophosphatidic acid, the G protein–linked neuropeptide receptors,
and growth factor receptors.64 PKC inhibitors
block tyrosine phosphorylation of
p125FAK in platelets bound to
immobilized IgG via a low-affinity receptor, FcRII, or
when the
IIbß3-integrin
receptor is engaged.30 No such inhibition was found after
stimulation of the G protein–linked neuropeptide
receptors.65 66 PKC-independent tyrosine
phosphorylation of p72syk has
also been observed during platelet aggregation stimulated by
fibrillar collagen.27 Previously, we found that rapid
adhesion to collagen under flow conditions primarily mediated by the
2ß1-integrin and in
the absence of
IIbß3-dependent
aggregation was associated with tyrosine
phosphorylation of
p125FAK.25 In the present study,
we show that rapid adhesion also resulted in tyrosine
phosphorylation of p72syk. We
also investigated a possible role of PKC activation in tyrosine
phosphorylation of p125FAK and
p72syk. PKC inhibitors did not modify
the adhesion-induced tyrosine phosphorylation of
p125FAK and p72syk (Figure 4), indicating that PKC is not required for
phosphorylation of these tyrosine kinases during
2ß1-integrin–mediated
adhesion. This result also indicates that different signaling pathways
are involved in tyrosine phosphorylation of
p125FAK during
IIbß3-mediated
aggregation, which is dependent on PKC activation,15 and
in the 2ß1-mediated
adhesion.
Because adhesion to polylysine caused strong phosphorylation of pleckstrin and soluble polylysine initiates a distinctive signal transduction via a specific receptor,67 it appears that polylysine might not provide an appropriate "nonspecific" control in studies of adhesion to ECM proteins. However, as far as the PKC dependence is concerned, mechanisms of adhesion to collagen and polylysine seem to be very different, and polylysine experiments provide a useful comparison to results with collagen. This may not be true for other ECM proteins.
PMA Stimulated Adhesion to Collagen
To complement our studies with PKC inhibitors, we
examined the effect of direct PKC stimulation with phorbol esters on
platelet adhesion and signaling events. We used 2 concentrations of
PMA, 20 and 100 nmol/L, which caused nearly equal pleckstrin
phosphorylation. This is consistent with
reports that pleckstrin phosphorylation reaches
saturation level (100%) at PMA concentrations >20
nmol/L.68 The lower concentration of PMA (20 nmol/L) had
no effect on rapid adhesion kinetics (Figure 7) and
adhesion-induced tyrosine phosphorylation of
p125FAK and p72syk. This
agrees with our findings with PKC inhibitors that PKC
activation evidenced by pleckstrin phosphorylation was
not required in the initial events of platelet adhesion to
collagen. However, the higher dose of PMA caused a significant
potentiation of adhesion as well as stimulation of tyrosine
phosphorylation of p125FAK and
p72syk. This PMA-stimulated adhesion was
independent of the formation of arachidonate metabolites,
released ADP, or RGD-dependent aggregation. The PKC
inhibitor GF 109203X completely blocked the PMA effect,
reducing enhanced adhesion.
These results show that PKC activation induced by 100 nmol/L PMA may regulate platelet adhesion, which at first sight seems to contradict our findings with PKC inhibitors, which exclude a regulatory role of PKC in initial rapid adhesion. However, it must be noted that PKC was activated much more by 100 nmol/L PMA than by adhesion alone (seen as increased pleckstrin phosphorylation) or by 20 nmol/L PMA. Different sensitivities to PKC inhibitors of control and 100 nmol/L PMA–stimulated adherent platelets also suggest that although PKC is activated in both situations, different degrees of activation (or different PKC isozymes) may be involved. Although we found that at higher PMA levels, PKC activation may regulate adhesion, such levels were not reached for unstimulated platelets in our adhesion assay (in the absence of secretion and aggregation). The results with unstimulated platelets and with platelets treated with 20 nmol/L PMA indicate that lower levels of PKC activity are not sufficient to influence rapid attachment. Therefore, it appears that adhesion of untreated platelets and platelets pretreated with 20 nmol/L PMA on the one hand and adhesion potentiation by 100 nmol/L PMA on the other involve 2 different mechanisms.
The precise mechanism by which PKC activation by 100 nmol/L PMA may
regulate adhesion is not yet known. It is unlikely that pleckstrin is
involved, because phosphorylation of pleckstrin did not
correlate with stimulation of adhesion (both 20 and 100 nmol/L PMA
induced the same level of pleckstrin phosphorylation,
yet adhesion was enhanced only at the higher dose). We found that 100
nmol/L (but not 20 nmol/L) PMA caused partial activation of
platelet ß1-integrin, as judged by an
increased exposure of the 15/7 epitope. The increased binding of the
15/7 antibody to platelets was not associated with upregulation of
the 2ß1-integrin,
because FACS analysis showed unchanged binding of the
anti-2 mAb P1E6 and
anti-ß1 mAb 1959 after PMA treatment. The
PMA-induced activation of platelet
ß1-integrin provides evidence of inside-out
signaling to the
2ß1-receptor and may
be directly responsible for the potentiation of adhesion. The
anti-2ß1 antibody 6F1
caused partial (60%) inhibition of the PMA-induced potentiation of
adhesion, most likely due to 6F1 sterically blocking the
collagen-binding sites on the ß1-integrin. This
suggests that the PMA stimulation is partially mediated by the
2ß1-receptor (most
likely as a result of ß1-activation), and the
remainder may be mediated via other receptors, such as
GPVI37 or GPIV.40
It is also possible that highly activated PKC, at levels greater than needed for saturation of pleckstrin phosphorylation, phosphorylates other regulators of adhesion. Indeed, PMA at higher concentrations (200 nmol/L) was found to stimulate PI3 kinase in platelets69 and potentiate arachidonic acid release in platelets activated with the ionophore A23187.68 We also observed that 100 nmol/L (but not 20 nmol/L) PMA induced phosphorylation of myosin light chain. This suggests that when PKC is highly activated, an increase in cytosolic calcium may influence adhesion.
Conclusions
We have found that platelet adhesion to collagen under flow
conditions causes rapid activation of PKC and tyrosine
phosphorylation of p125FAK and
p72syk. PKC inhibitors had no effect
on basic adhesion kinetics and adhesion-induced tyrosine
phosphorylation of p125FAK and
p72syk, suggesting that PKC activity is not
required in the initial events of adhesion and tyrosine
phosphorylations. In contrast, the PKC
inhibitor GF 109203X destabilized adherent platelets
after initial attachment. This resulted in accelerated detachment of
the adherent platelets from the collagen surface, indicating that
PKC activity is necessary for stabilizing focal contacts between
platelet collagen receptors and cytoskeletal structures in the
later stages of platelet adhesion. In addition, strong PKC
activation induced by 100 nmol/L PMA markedly potentiated the rapid
early phase of adhesion to collagen and was associated with a
conformational change in ß1-integrins detected
as an increased exposure of the 15/7 epitope on platelets. This
suggests that a high level of PKC activation in platelets initiates
"inside-out" signaling, leading to activation of
ß1-integrins and to subsequent stimulation of
the
2ß1-integrin-dependent
adhesion.
|
Acknowledgments |
|---|
This work was supported by a grant from the Carman Trust and for the original equipment by a grant from the NIH, NHLB1-27014. We would like to thank Linda Beggerly for blood drawing, Brandon Hunter for technical assistance, William Ross for FACS analysis, and Carl Simon for useful discussion.
Received January 19, 1999; accepted July 8, 1999.
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References |
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